Glucoamylase engineered yeast and fermentation methods

ABSTRACT

The invention is directed to an engineered yeast including an exogenous nucleic acid encoding a glucoamylase comprising SEQ ID NO:1 and SEQ ID NO:4, or a variant thereof. The engineered yeast are able to provide glucoamylase into a fermentation media and cause degradation of starch material generating glucose for fermentation to a desired bioproduct, such as ethanol. High titers of bioproduct (e.g., 70 g/kg of ethanol) can be achieved, along with low residual glucose levels. Further the yeast exhibit good growth and bioproduct product at temperatures of 32° C. or greater.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/636,716, filed Feb. 28, 2018, and entitled“GLUCOAMYLASE ENGINEERED YEAST AND FERMENTATION METHODS”, whichapplication is hereby incorporated by reference herein in its entirety.

SEQUENCE LISTING

The entire contents of the ASCII text file entitled“N00570_Sequence_Listing_ST25.txt,” created on Feb. 27, 2019, and havinga size of 123 kilobytes is incorporated herein by reference.

FIELD OF THE INVENTION

The current invention relates to yeast engineered with exogenousglucoamylase nucleic acids and fermentations methods for producing abioproduct such as ethanol.

BACKGROUND

Many fermentation feedstocks are derived from plant sources (e.g., cornmash) where the carbohydrates are predominantly in the form of starchpolymers. The starch polymers in such feedstocks must be treated to lowmolecular weight sugars that can be consumed by the yeast and used forgrowth and bioproduct production. Typical treatments include acid and/orenzymatic hydrolysis where the polymer chain is hydrolyzed to generatethe sugars that can be used by the yeast. Starch degrading enzymes suchas alpha amylases and glucoamylases can be added to convert the polymerto simple sugars. However, such enzyme additions can add significantcost and complexity to the fermentation process.

Heterologous expression and functionality of enzymes in yeast to aid instarch hydrolysis can be challenging, as it is difficult to know if thenucleic acid will be expressed properly and a functional enzyme willform, and if an active form of the enzyme will be secreted from thecell. It is also challenging to engineer yeast for growth and bioproductproduction at non-optimal conditions, such as high temperatures, and inhigh bioproduct titers. For example, while ethanol production byfermentation is a well know industrial process, maintaining ethanolrates, titers, and yields while at the same time engineering the yeastto reduce reliance on supplemental enzymes, growth under non-optimalconditions (e.g., temperature), and minimizing by-product formation canbe technically difficult. Increased ethanol concentration andaccumulation of undesirable byproducts can also be detrimental to cellhealth.

SUMMARY OF THE INVENTION

The invention relates to engineered yeast and fermentation methods,wherein the engineered yeast are able to secrete a heterologousglucoamylase (GA) into a fermentation medium and provide glucoamylaseactivity (E.C. 3.2.1.3) on a fermentation substrate. The invention alsorelates to methods of for producing bio-derived products, such asethanol, via fermentation using the engineered yeast.

In one aspect, experimental studies associated with the currentapplication identified fungal glucoamylase genes that, when introducedexogenously into yeast, allowed it to grow well on feedstocks containinglow glucose and high starch amounts, and produce high levels ofbioproduct. The results indicated that the engineered yeast were able tosecrete glucoamylase into the fermentation medium and that theglucoamylase was enzymatically active towards the starch to generatesufficient glucose for growth and bioproduct production. Other benefitsassociated with the disclosure include improved fermentation andbioproduct production at elevated fermentation temperatures. Yet otherbenefits associated with the disclosure include reduced amounts ofglucose at the end of the fermentation period.

In one aspect, the invention provides an engineered yeast comprising anexogenous nucleic acid encoding a glucoamylase comprising a sequencehaving 81% or greater sequence identity to SEQ ID NO:1 (Rhizopusmicrosporus glucoamylase). In an embodiment, the engineered yeast iscapable of producing ethanol at a rate of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, or 5.0 g/L*h or greater during a fermentation process. Inanother embodiment, the engineered yeast is capable of producing (a) atleast 70 g/kg of ethanol in a fermentation medium made from a glucosepolymer-containing feedstock having (i) a DE of about 50 or less. Inanother embodiment, the engineered yeast is capable of producing (a) 90g/kg or greater, 120 g/kg or greater, 130 g/kg or greater, or 140 g/kgor greater of ethanol in a fermentation medium made from a glucosepolymer-containing feedstock having (i) a DE of about 30. Inembodiments, the amount of 70 g/kg of ethanol may be produced within 48hours of inoculation in a fermentation medium with the feedstock. Inembodiments, the glucose concentration may not be greater than 5% (wt)in the fermentation medium at the beginning (inoculation) of thefermentation process. In embodiments, the feedstock may provide anamount of glucose polymer-containing feedstock sufficient to produce 70g/kg ethanol, for example about 20 wt % glucose-polymer feedstock in themedium. In embodiments, the feedstock may have one or more of thefollowing properties: the feedstock is a starch hydrolysate; theglucose-polymer has predominantly α-1,4 linkages; the feedstocksubstantially excludes cellulosic materials (e.g., less than 20%, 15%,or 10% of cellulosic material).

In an embodiment, the yeast is capable of producing (a) at least 70 g/kgof ethanol in a fermentation medium made from corn mash having a DE of30±2, wherein fermentation medium comprises about 32% dry weight ofcorn, and a pH 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin,wherein the staring yeast concentration is 0.1 (OD600), and fermentationis carried out at 48 hrs at 30° C. with agitation. In more specificembodiments that engineered yeast comprise a glucoamylase having 81%,82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequenceidentity to SEQ ID NO:1.

In a related embodiment, the invention provides a fermentation methodfor producing a bioproduct. The method comprises forming a fermentationmedium from a glucose polymer-containing feedstock, and then fermentingthe fermentation medium using an engineered yeast comprising anexogenous nucleic acid encoding a glucoamylase comprising a sequencehaving 81% or greater sequence identity to SEQ ID NO:1. Fermentation iscarried out over a period to produces the bioproduct, such as ethanol.

In more specific embodiments, ethanol is produced during thefermentation period to an amount of 70 g/kg or greater, 90 g/kg orgreater, 120 g/kg or greater, 130 g/kg or greater, or 140 g/kg orgreater, in the fermentation medium. In some embodiments, in thefermentation method, the glucose polymer-containing feedstock has adextrose equivalent (DE) that is not greater than 50. In someembodiments, glucose is not greater than 5 wt % of solids materials inthe feedstock. Optionally, supplemental glucoamylase can be introducedinto the fermentation methods to increase bioproduct titers.

In some embodiments using the engineered yeast with glucoamylase having81% or greater sequence identity to SEQ ID NO:1 fermenting is carriedout at a temperature in the range of 31° C. to 35° C. for most or all ofa fermentation period.

In some embodiments using the engineered yeast with glucoamylase having81% or greater sequence identity to SEQ ID NO:1, at the end of thefermentation period the glucose concentration in the fermentation mediumof not greater than 1.0 g/L.

In another aspect, the invention provides an engineered yeast comprisingan exogenous nucleic acid encoding a glucoamylase comprising a sequencehaving 97% or greater, or 98% or greater sequence identity to SEQ IDNO:4 (Rhizopus delemar glucoamylase). In an embodiment, the engineeredyeast is capable of producing ethanol at a rate of 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, or 5.0 g/L*h or greater during a fermentationprocess. In another embodiment, the engineered yeast is capable ofproducing (a) at least 70 g/kg of ethanol in a fermentation medium madefrom a glucose polymer-containing feedstock having (i) a DE of about 30.In embodiments, the amount of 70 g/kg of ethanol may be produced within48 hours of inoculation in a fermentation medium with the feedstockhaving. In embodiments, the glucose concentration may not be greaterthan 5% (wt) in the fermentation medium at the beginning (inoculation)of the fermentation process. In embodiments, the feedstock may providean amount of glucose polymer-containing feedstock sufficient to produce70 g/kg ethanol, for example about 20 wt % glucose-polymer feedstock inthe medium. In embodiments, the feedstock may have one or more of thefollowing properties: the feedstock is a starch hydrolysate; theglucose-polymer has predominantly α-1,4 linkages; the feedstocksubstantially excludes cellulosic materials (e.g., less than 20%, 15%,or 10% of cellulosic material).

In a related embodiment, the invention provides a fermentation methodfor producing a bioproduct. The method comprises forming a fermentationmedium from a glucose polymer-containing feedstock, and then fermentingthe fermentation medium using an engineered yeast comprising anexogenous nucleic acid encoding a glucoamylase comprising a sequencehaving 97% or greater sequence identity to SEQ ID NO:4. Fermentation iscarried out over a period to produces the bioproduct, such as ethanol.

In more specific embodiments of any of the preceding fermentation,ethanol is produced during the fermentation period to an amount of 70g/kg or greater, 90 g/kg or greater, 120 g/kg or greater, 130 g/kg orgreater, or 140 g/kg or greater, in the fermentation medium. In someembodiments, in the fermentation method, the glucose polymer-containingfeedstock has a dextrose equivalent (DE) that is about 30. In someembodiments, glucose is not greater than 5 wt % of solids materials inthe feedstock. Optionally, supplemental glucoamylase can be introducedinto the fermentation methods to increase bioproduct titers.

In some embodiments using the engineered yeast with glucoamylase having97% or greater or 98% or greater sequence identity to SEQ ID NO:4,fermenting is carried out at a temperature in the range of 31° C. to 35°C. for most or all of a fermentation period.

In some embodiments using the engineered yeast with glucoamylase having97% or greater sequence identity to SEQ ID NO:4, at the end of thefermentation period the glucose concentration in the fermentation mediumis not greater than 2.0 L, or not greater than 1.0 g/L.

In more specific embodiments of any of the preceding yeast embodiments,the engineered yeast is capable of producing 90 g/kg or greater, 120g/kg or greater, 130 g/kg or greater, or 140 g/kg or greater of ethanolin a fermentation medium made from a glucose polymer-containingfeedstock having (i) a DE of about 30.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows an alignment of SEQ ID NO:1 to Rhizopus oryzae GA, showingthe signal sequence (1-25) and starching binding domain (26-131).

DETAILED DESCRIPTION

The aspects of the present invention described below are not intended tobe exhaustive or to limit the invention to the precise forms disclosedin the following detailed description. Rather a purpose of the aspectschosen and described is so that the appreciation and understanding byothers skilled in the art of the principles and practices of the presentinvention can be facilitated.

An aspect of the invention relates to engineered yeast that expresses aglucoamylase comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%,90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to SEQ IDNO:1 (Rhizopus microsporus glucoamylase (GA)). Another aspect of theinvention relates to engineered yeast that expresses a glucoamylasecomprising a sequence having 97%, 98%, or 99% or greater sequenceidentity to SEQ ID NO:4 (Rhizopus delemar glucoamylase (GA)). Engineeredyeast of the disclosure are able express and provide glucoamylase enzymein the culture medium, and the glucoamylases are enzymatically active onglucose polymer substrates such as starch from various plant sources.The glucoamylase activity within the medium can generate mono anddisaccharide sugars which can be consumed by the yeast and can be usedas a carbon source for the production of a target compound, such asethanol.

In embodiments of the disclosure, a fermentation medium can be preparedfrom a feedstock having glucose polymer and minimal glucose. Forexample, a fermentation medium can be prepared from a starch-containingfeedstock having a DE (dextrose equivalent) of 70 or less, 60 or less,50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less,or 5 or less. Dextrose equivalent (DE) is a measure of the amount ofreducing sugars present in a material (e.g., a sugar product or astarch-containing feedstock), relative to dextrose (a.k.a. glucose),expressed as a percentage on a dry basis. For example, a maltodextrinwith a DE of 10 would have 10% of the reducing power of dextrose (whichhas a DE of 100). A fermentation medium can be prepared from astarch-containing feedstock that does not have a glucose amount that isgreater than about 5% wt., or greater than about 4% wt., or greater thanabout 3% wt., or greater than about 2% wt., or greater than about 1%wt., per total solids in the feedstock. In some fermentation methods thelow glucose-containing starch feed stock is added periodically orcontinuously throughout the fermentation period. The glucoamylasesproduced by the engineered yeast can be enzymatically active against thestarch in the medium and generate glucose which can be used by the yeastfor growth and generation of bioproduct. Optionally, the fermentationmethod can include supplementing the medium with purified glucoamylase,such as glucoamylase obtained from a commercial source, which canfurther drive enzymatic hydrolysis of the starch and increase growth andtiters of bioproduct.

For example, without any commercial glucoamylase enzyme supplementationand using a low glucose feedstock, the engineered yeast of thedisclosure can generate an amount of ethanol of about 70 g/kg orgreater, such as an amount in the range of about 70 g/kg to about 115g/kg, about 75 g/kg to about 115 g/kg, about 80 g/kg to about 115 g/kg,about 85 g/kg to about 115 g/kg, about 90 g/kg to about 115 g/kg, about95 g/kg to about 115 g/kg, about 100 g/kg to about 115 g/kg, or about105 g/kg to about 115 g/kg of ethanol in the fermentation medium. Inembodiments wherein supplemental glucoamylase is added to the medium,greater amounts of ethanol can be produced, such as an amount of 110g/kg or greater, or 125 g/kg or greater, or 140 g/kg or greater, in thefermentation medium.

In further embodiments, yeast engineered with glucoamylases of thedisclosure can exhibit excellent fermentation performance attemperatures greater than standardly used in fermentations (i.e.,fermentations at 30° C. using Saccharomyces cerevisiae as a hostorganism). For example, in some embodiments using the engineered yeastof the disclosure, fermenting is carried out at a temperature in therange of 31° C. to 35° C., or 32° C. to 34° C., for most or all of afermentation period. Even at the higher temperatures, the engineeredyeast are able to generate glucoamylase activity in the medium, andpromote excellent cell growth and bioproduct production.

In further embodiments, following a period of fermentation, yeastengineered with of glucoamylases of the disclosure can provide adesirable final fermentation medium with high levels of bioproduct andlow levels of byproduct. In particular, the final fermentation mediumcan have high levels of ethanol (e.g., 70 g/kg or greater), understressful fermentation conditions, such as where low levels of glucose,such as 1.0 g/kg, may be present.

The term “exogenous” as used herein, means that a molecule, such as anucleic acid, or an activity, such as an enzyme activity, is introducedinto the host organism. An exogenous nucleic acid can be introduced into the host organism by well-known techniques and can be maintainedexternal to the hosts chromosomal material (e.g., maintained on anon-integrating vector), or can be integrated into the host'schromosome, such as by a recombination event. An exogenous nucleic acidcan encode an enzyme, or portion thereof, that is either homologous orheterologous to the host organism.

The term “heterologous” refers to a molecule or activity that is from asource that is different than the referenced molecule or organism.Accordingly, a gene or protein that is heterologous to a referencedorganism is a gene or protein not found in that organism. For example, aspecific glucoamylase gene found in a first fungal species andexogenously introduced into a second fugal species that is the hostorganism is “heterologous” to the second fungal organism.

The following SEQ ID NOs are associated with the fungal GA amino acid orprotein sequences: SEQ ID NO:1: Rhizopus microsporus GA amino acidsequence; SEQ ID NO:2: Rhizopus microsporus GA nucleic acid sequence #1;SEQ ID NO:3: Rhizopus microsporus GA nucleic acid sequence #2; SEQ IDNO:4: Rhizopus delemar GA amino acid sequence; SEQ ID NO:5: Rhizopusdelemar GA nucleic acid sequence #1; and SEQ ID NO:6: Rhizopus delemarGA nucleic acid sequence #2.

Table 1 is a table of sequence identity (global protein alignment)between the Rhizopus microsporus GA amino acid sequence and GA sequenceof other known GAs. The reference molecule is Rhizopus microsporus GA,and scoring matrix was BLOSUM 62.

TABLE 1 GA # # % sequence Start End Match Nonmatch Match Rhizopusmicrosporus 1 605 — — 100 Rhizopus delemar 1 604 488 117 80 Rhizopusoryzae 1 604 485 120 80 Mucor ambiguus 1 609 416 194 68 Mucor circenello1 609 415 198 67 Choanephora cucurbitarum 1 622 402 222 64 Phycomycesblakesleeanus 1 598 369 243 60 Arthrobotrys oligospora 1 621 204 441 31

Rhizopus microsporus GA and Rhizopus delemar GA are members of theglucoamylase enzyme family (E.C. 3.2.1.3) and are amylolytic enzymesthat hydrolyze 1,4-linked a-D-glucosyl residues successively from thenonreducing end of oligo- and polysaccharide chains with the release ofD-glucose. Alternative names for glucoamylases include amyloglucosidase;γ-amylase; lysosomal α-glucosidase; acid maltase; exo-1,4-α-glucosidase;glucose amylase; γ-1,4-glucan glucohydrolase; acid maltase;1,4-α-D-glucan glucohydrolase.

Glucoamylases such as Rhizopus microsporus GA and Rhizopus delemar GAcan also cleave α-1,6 bonds on amylopectin branching points. As usedherein, the term “amylolytic activity” pertains to these enzymaticmechanisms.

Engineered yeast of the disclosure can include variant(s) of the naturalsequences of the Rhizopus microsporus GA and Rhizopus delemar GAglucoamylase polypeptide and can include one or more amino acidvariations, providing for a non-natural polypeptide. Polypeptides of thedisclosure can be a portion of the naturally occurring Rhizopusmicrosporus GA and Rhizopus delemar GA sequence (such as polypeptidesthat are truncated at its N-terminus, its C-terminus, or both), whilethe glucoamylase polypeptide retains amylolytic activity.

N-terminal truncations can be produced by altering the position of theATG start codon, while ensuring the sequence downstream remains inframe. C-terminal variants can be produced by inserting an in-framepremature stop codon. Random methods such as error-prone PCR could alsobe employed, and could be combined with growth on starch to ensurepeptide function.

Variations in the Rhizopus microsporus GA (SEQ ID NO:1) and Rhizopusdelemar GA (SEQ ID NO:4) sequences can be made with information aboutthe primary sequence of these enzymes, through sequence alignments, andin view of information regarding other glucoamylase enzymes as known inthe art. Most glucoamylases, including Rhizopus microsporus GA andRhizopus delemar GA, are multidomain enzymes. Many glucoamylases,including Rhizopus microsporus GA and Rhizopus delemar GA, include astarch-binding domain connected to a catalytic domain via anO-glycosylated linker region, based on known crystal structures fromsimilar enzymes.

Glucoamylases may also have a catalytic domain having a configuration ofa configured twisted (alpha/alpha)(6)-barrel with a centralfunnel-shaped active site. Glucoamylases may have a structurallyconserved catalytic domain of approximately 450 residues. In someglucoamylases the catalytic domain generally followed by a linker regionconsisting of between 30 and 80 residues that are connected to a starchbinding domain of approximately 100 residues.

Glucoamylase properties may be correlated with their structuralfeatures. A structure-based multisequence alignment was constructedusing information from catalytic and starch-binding domain models (see,e.g., Coutinho, P. M., and Reilly, P. J., 1994. Protein Eng. 7:393-400and 749-760). It has been shown that the catalytic and starch bindingdomains are functionally independent based on structure-functionrelationship studies, and there are structural similarities in microbialglucoamylases. From other studies, specific glucoamylase residues havebeen shown to be involved in directing protein conformational changes,substrate binding, thermal stability, and catalytic activity (see, forexample, Sierks, M. R., et al. 1993. Protein Eng. 6:75-79; and Sierks,M. R., and Svensson, B. 1993. Biochemistry 32:1113-1117).

Therefore, the correlation between glucoamylase sequence and proteinfunction is understood in the art, and one of skill could design andexpress variants of amylolytically active glucoamylases having one ormore amino acid deletion(s), substitution(s), and/or additions. Forexample, in some aspects, the glucoamylase portion of the Rhizopusmicrosporus GA and Rhizopus delemar GA can contain a truncated versionof a naturally occurring glucoamylase, the truncated version having, inthe least, a catalytic and optionally a starch-binding domain havingamylolytic activity as described herein.

Truncated forms of glucoamylase have been generated and have been shownto have enzymatic activity. For example, Evans et al. (Gene, 91:131;1990) generated a series of truncated forms of glucoamylase toinvestigate how much of the O-glycosylated region was necessary for theactivity or stability of GAIL a fully active form of the enzyme lackingthe raw starch-binding domain. It was found that a significant portionof the C-terminus could be deleted from GAII with insignificant effecton activity, thermal stability, or secretion of the enzyme.

Lin et. al (BMC Biochemistry 8:9, 2007) teaches there was no loss ofglucoamylase activity the starch binding domain located betweenpositions 26-131 in the Rhizopus oryzae glucoamylase was deleted. Also,Mertens & Skory (Enz. Microbiol. Technology 40: 874-880, 2007) isolateda natural glucoamylase which lacked a starch binding domain.

Various amino acids substitutions associated with causing a change inglucoamylase activity are also known in the art. Substitution(s) ofamino acid(s) at various locations in the glucoamylase sequence havebeen shown to affect properties such as thermo stability, starchhydrolysis activity, substrate usage, and protease resistance. As such,the current disclosure contemplates use of a Rhizopus microsporus GA andRhizopus delemar GA sequence that includes one or more amino acidssubstitution(s) in the glucoamylase portion of the polypeptide, whereinthe substitutions differ from the wild type sequence of theglucoamylase.

For example, U.S. Pat. No. 8,809,023 describes a method for reducing theratio between isomaltose synthesis and starch hydrolysis activity (IS/SHratio) during the hydrolysis of starch. In particular, a Trichodermareesei glucoamylase (Tr GA) is described (total length of 632 aminoacids having an N-terminal having a signal peptide) that is modified atwith amino acid positions as follows: D44R and A539R; or D44R, N61I, andA539R. This glucoamylase variant is reported to exhibit a reduced IS/SHratio compared to said parent glucoamylase during the hydrolysis ofstarch.

As another example, U.S. Pat. No. 8,592,194 describes glucoamylasevariants with increased thermo stability compared to wild typeglucoamylase variants. Also described in this disclosure is theTrichoderma reesei glucoamylase but instead one or more amino acidsubstitutions to the native Tr GA sequence at positions 10, 14, 15, 23,42, 45, 46, 59, 60, 61, 67, 68, 72, 73, 97, 98, 99, 102, 108, 110, 113,114, 122, 124, 125, 133, 140, 144, 145, 147, 152, 153, 164, 175, 182,204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 240, 241, 242,244, 263, 264, 265, 268, 269, 276, 284, 291, 300, 301, 303, 310, 311,313, 316, 338, 342, 344, 346, 349, 359, 361, 364, 379, 382, 390, 391,393, 394, 408, 410, 415, 417, and 418. As an example, the currentdisclosure contemplates creating variants at amino acid locations in SEQID NO:1 and SEQ ID NO:4 corresponding to the respective describedpositions in the TrGA sequence in order to provide variants withincreased thermostability.

The determination of “corresponding” amino acids from two or moreglucoamylases can be determined by alignments of all or portions oftheir amino acid sequences. Sequence alignment and generation ofsequence identity include global alignments and local alignments, whichtypically use computational approaches. In order to provide globalalignment, global optimization forcing sequence alignment spanning theentire length of all query sequences is used. By comparison, in localalignment, shorter regions of similarity within long sequences areidentified.

As used herein, an “equivalent position” means a position that is commonto the two sequences (e.g., a template GA sequence and a GA sequencehaving the desired substitution(s)) that is based on an alignment of theamino acid sequences of one glucoamylase or as alignment of thethree-dimensional structures. Thus, either sequence alignment orstructural alignment, or both, may be used to determine equivalence.

In some modes of practice, the BLAST algorithm is used to compare anddetermine sequence similarity or identity. In addition, the presence orsignificance of gaps in the sequence which can be assigned a weight orscore can be determined. These algorithms can also be used fordetermining nucleotide sequence similarity or identity. Parameters todetermine relatedness are computed based on art known methods forcalculating statistical similarity and the significance of the matchdetermined. Gene products that are related are expected to have a highsimilarity, such as greater than 50% sequence identity. Exemplaryparameters for determining relatedness of two or more sequences usingthe BLAST algorithm can be as follows.

Inspection of nucleic acid or amino acid sequences for two nucleic acidsor two polypeptides will reveal sequence identity and similaritiesbetween the compared sequences. Sequence alignment and generation ofsequence identity include global alignments and local alignments whichare carried out using computational approaches. An alignment can beperformed using BLAST (National Center for Biological Information (NCBI)Basic Local Alignment Search Tool) version 2.2.31 software with defaultparameters. Amino acid % sequence identity between amino acid sequencescan be determined using standard protein BLAST with the followingdefault parameters: Max target sequences: 100; Short queries:Automatically adjust parameters for short input sequences; Expectthreshold: 10; Word size: 6; Max matches in a query range: 0; Matrix:BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositionaladjustments: Conditional compositional score matrix adjustment; Filter:none selected; Mask: none selected. Nucleic acid % sequence identitybetween nucleic acid sequences can be determined using standardnucleotide BLAST with the following default parameters: Max targetsequences: 100; Short queries: Automatically adjust parameters for shortinput sequences; Expect threshold: 10; Word size: 28; Max matches in aquery range: 0; Match/Mismatch Scores: 1, −2; Gap costs: Linear; Filter:Low complexity regions; Mask: Mask for lookup table only. A sequencehaving an identity score of XX % (for example, 80%) with regard to areference sequence using the NCBI BLAST version 2.2.31 algorithm withdefault parameters is considered to be at least XX % identical or,equivalently, have XX % sequence identity to the reference sequence.

A global alignment can align sequences with significant identity to, forexample, the SEQ ID NO:1 (Rhizopus microsporus GA) or SEQ ID NO:4Rhizopus delemar GA glucoamylase in order to determine whichcorresponding amino acid position(s) in the target sequence (e.g., aglucoamylase ortholog) can be substituted with the one or more of theamino acid if a glucoamylase variant is used.

In some cases, the substitution can be a conservative substitution, suchas where one amino acid of a particular type (e.g., polar,non-polar/aliphatic, positively charged/basic, negativelycharged/acidic) is replaced with an amino acid of the same type.Exemplary conservative amino acid substitutions of the presentdisclosure can involve exchange of one aliphatic or hydrophobic aminoacid Ala, Val, Leu, or Ile for another; exchange of one hydroxyl aminoacid Ser or Thr for the other; exchange of one acidic amino acid Asp orGlu for the other; exchange of one amide amino acid Asn or Gln for theother, exchange of one basic amino acid Lys, Arg, for His for another;exchange of one aromatic amino acid Phe, Tyr, or Trp, for another, andexchange of one small amino acids Ala, Ser, Thr, Met, or Gly foranother.

In embodiments of the disclosure, SEQ ID NO:1 has one or more amino acidmutations which causes it to be less than 100% identical to SEQ ID NO:1.For example, the glucoamylase may have multiple amino acid deletion(s),substitution(s), and/or additions causing it to have about 81% orgreater identity to SEQ ID NO:1, 82% or greater, 83% or greater, 84% orgreater, 85% or greater, 86% or greater, 87% or greater, 88% or greater,89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% orgreater, 94% or greater, 95% or greater, 96% or greater, 97% or greater,98% or greater, or 99% or greater identity to SEQ ID NO:1. A variantwith a single amino acid substitution has 99.8% identity to SEQ ID NO:1.

In exemplary embodiments, more than one location in SEQ ID NO:1 can bechanged to provide a variant that has to SEQ ID NO:1 that is less than80%. For example, changes to the signal sequence and deletion of thestarch binding domain can provide a variant with less than 80% identityto SEQ ID NO:1, such as about 75%-80% identity to SEQ ID NO:1.

FIG. 1 shows an alignment of SEQ ID NO:1 to Rhizopus oryzae GA, showingthe signal sequence (1-25) and starching binding domain (26-131).

Table 2 is a table of sequence identity (BLAST alignment) of a “core”sequence of SEQ ID NO:1 (lacking signal sequence and starch bindingdomain) GA sequences of other known GAs. Accession CEG69155.1 is thesame sequence as SEQ ID NO:1.

TABLE 2 Source Accession # Start End Match NonMatch % Match Rhizopusmicrosporus CEG69155.1 132 605 Rhizopus microsporus ORE14155.1 132 605458 16 96 Rhizopus delemar EIE75378.1 132 604 397 77 83 Rhizopus oryzaeP07683.2 132 604 394 80 83 Rhizopus oryzae BAH09876.1 132 604 395 79 83Rhizopus oryzae ABB77799.1 132 604 396 78 83

In embodiments of the disclosure, SEQ ID NO:4 has one or more amino acidmutations which causes it to be less than 100% identical to SEQ ID NO:4.For example, the glucoamylase may have multiple amino acid deletion(s),substitution(s), and/or additions causing it to have about 98% orgreater, or 99% or greater identity to SEQ ID NO:4. A variant with asingle amino acid substitution has 99.8% identity to SEQ ID NO:4.

In exemplary embodiments, one or more of locations in SEQ ID NO:1 or SEQID NO:4 are changed to provide a variant SEQ ID NO:1 and SEQ ID NO:4also generally include a native “signal sequence.” Various other termsmay be used to indicate a “signal sequence” as known in the art, such aswhere the word “signal” is replaced with “secretion” or “targeting” or“localization” or “transit” or leader,” and the word “sequence” isreplaced with “peptide” or “signal.” Generally, a signal sequence is ashort amino acid stretch (typically in the range of 5-30 amino acids inlength) that is located at the amino terminus of a newly synthesizedprotein. Most signal peptides include a basic N-terminal region(n-region), a central hydrophobic region (h-region) and a polarC-terminal region (c-region) (e.g., see von Heijne, G. (1986) NucleicAcids Res. 14, 4683-4690).

In SEQ ID NO:1 and SEQ ID NO:4 the predicted signal sequence is fromamino acid 1 to 25 of SEQ ID NO:1 and from amino acid 1 to 25 of SEQ IDNO:4, respectively. A signal sequence can target the protein to acertain part of the cell, or can target the protein for secretion fromthe cell. For example, it has been shown that the native N-terminalsignal sequence of the S. diastaticus Glucoamylase STAI gene can targetit to the endoplasmic reticulum of the secretory apparatus (for example,see Yamashita, I. et al., (1985) J. Bacteriol. 161, 567-573).

Glucoamylase enzymes of the disclosure can use the native signalsequences of SEQ ID NO:1 and SEQ ID NO:4, or variants thereof, or can bemodified to include a heterologous signal sequences. In one aspect, thecurrent invention provides the partial or full replacement of the nativesignal sequence of SEQ ID NO:1 and SEQ ID NO:4 with a secretion signalbased on a sequence at the N-terminal portion of An aa, Sc FAKS, Sc AKS,Sc MFα1, Sc IV, Gg LZ, and Hs SA as described in U.S. Provisional PatentApplication 62/371,681 (published as WO2018027131) and PCT ApplicationNo. PCT/US2016/016822 (published as WO2016127083), both of which arehereby incorporated by reference in their entirety.

These secretion signals can be used as a replacement to the nativesecretion signal of the SEQ ID NO:1 and SEQ ID NO:4, or can be used inaddition to the native secretion signal. In view of the addition of theheterologous secretion signal, the proteins may be referred to as“fusion proteins,” and annotated as follows: [An aa-SS]-[Rm GA], [ScIV-SS]-[Rd GA], etc.

Possible heterologous N-terminal replacement sequences for theN-terminal of SEQ ID NO:1 and SEQ ID NO:4 include the following. Sc-FAKSis a sequence of 90 amino acids derived from the N-terminal portion ofthe Saccharomyces cerevisiae peptide mating pheromone α-factor (e.g.,see Brake, A., et al., Proc. Natl. Acad. Sci., 81:4642-4646, 1984;Kurjan, J. & Herskowitz, I., Cell 30:933-943, 1982). Sc-MFα1 is aminoacids 20-89 Sc-FAKS. Sc IV a 19 amino acid N-terminal signal peptide ofa sucrose hydrolase enzyme (e.g, see, Carlson M., et al. (1983) Mol.Cell. Biol. 3:439-447). Gg LZ (also known as egg white lysozyme) is an18 amino acid N-terminal signal peptide of a glycoside hydrolase enzyme(e.g, see, Jigami et al. (1986) Gene 43:273-279). Hs SA is an 18 aminoacid N-terminal signal peptide of a serum (e.g, see, Kober et al. (2013)Biotechnology and Bioengineering; 110:1164-1173.). Sc MFα2 is derivedfrom the N-terminus the Saccharomyces cerevisiae mating factor alpha 2gene (Sc MFα2). Sc PHOS is derived from the N-terminus of theSaccharomyces cerevisiae repressible acid phosphatase (Meyhack et al.,EMBO J. 6:675-680, 1982).

Molecular techniques can be performed to create a nucleic acid sequencethat is a template for the expression of genes encoding SEQ ID NO:1 orSEQ ID NO:4, or variants thereof. As a general matter, a nucleic acid isprepared to encode a protein comprising SEQ ID NO:1 or SEQ ID NO:4, orvariants thereof.

In other aspects, the SEQ ID NO:1 or SEQ ID NO:4, or variants thereofoptionally comprises additional sequence that is not present in thenative glucoamylase polypeptide. The additional sequence, in someaspects, can provide functionality to the glucoamylase that is notpresent in the native polypeptide. Additional functionalities include,for example, protease sites or binding sites for other proteins ormaterials, or linker regions.

Nucleic acids sequences encoding SEQ ID NO:1 or SEQ ID NO:4, or variantsthereof, as well as any regulatory sequence (e.g., terminator, promoter,etc.) and vector sequence (e.g., including a selection marker,integration marker, replication sequence, etc.) can, in some modes ofpractice, be prepared using known molecular techniques. General guidancefor methods for preparing DNA constructs (e.g., for the DNA constructsincluding nucleic acids encoding SEQ ID NO:1 or SEQ ID NO:4 or a variantthereof) can be found in Sambrook et al Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989; and Ausubel et al. Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York, N.Y., 1993. PCR techniquescan be used for modifying nucleic acids encoding SEQ ID NO:1 or SEQ IDNO:4, to optionally introduce one or more mutations in the sequence toprovide a variant.

PCR techniques are described in, for example, Higuchi, (1990) in PCRProtocols, pp. 177-183, Academic Press; Ito et al (1991) Gene 102:67-70;Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al(1989) Nuc. Acids Res. 17:723-733. The techniques may optionally includesite-directed (or oligonucleotide-mediated) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared DNAencoding a glucoamylase polypeptide.

Alternatively, nucleic acid molecules can be generated by custom genesynthesis providers such as ATUM (Menlo Park, Calif.) or GeneArt (LifeTechnologies, Thermo Fisher Scientific).

An expression vector can be constructed to include the glucoamylasenucleic acid sequence operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the host organisms include, for example, plasmids, episomes andartificial chromosomes. The vectors can include selection sequences ormarkers operable for stable integration into a host chromosome.Additionally, the vectors can include one or more selectable markergenes and appropriate expression control sequences. Selectable markergenes also can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture medium. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art.

In some aspects, the nucleic acid can be codon optimized. The nucleicacid template can be the native DNA sequence that codes for theglucoamylase, or the template can be a codon-optimized version that isoptimized for expression in a desired host cell. Databases that provideinformation on desired codon uses in particular host organisms are knownin the art.

The DNA construct comprising the glucoamylase nucleic acid can beoperably linked to a promoter sequence, wherein the promoter sequence isfunctional in a host cell of choice. In some aspects, the promoter showstranscriptional activity in a fungal host cell and may be derived fromgenes encoding proteins either homologous or heterologous to the hostcell. In some aspects, the promoter is useful for expression in S.cerevisiae. Examples of well-known constitutive promoters include, butare not limited to the cytochrome c promoter (pCYC), translationalelongation factor promoter (pTEF), the glyceraldehyde-3-phosphatedehydrogenase promoter (pGPD/TDH3), the phosphoglycerate kinase promoter(PGK), and the alcohol dehydrogenase promoter (pADH). Optionally, anadditional factor that controls expression such as an enhancer or thelike may also be included on the vector.

The expression vector including the glucoamylase gene can also includeany termination sequence functional in the host cell. For example, thetermination sequence and the promoter sequence can be from the samecell, or the termination sequence is homologous to the host cell. Thetermination sequence can correspond to any promoter that is used.

The DNA construct may be introduced into a host cell using a vector. Thevector may be any vector which when introduced into a host cell isstably introduced. In some aspects, the vector is integrated into thehost cell genome and is replicated. Vectors include cloning vectors,expression vectors, shuttle vectors, plasmids, phage particles,cassettes and the like. In some aspects, the vector is an expressionvector that comprises regulatory sequences operably linked to theglucoamylase coding sequence. SEQ ID NOs as described herein can beassembled in the cell by the transformation of multiple smaller DNAfragments (e.g., “SEQ ID NO sub-fragments”) with overlapping homologythat in total constitute a particular SEQ ID NO. For example, theintegration of a desired SEQ ID NO, or portion thereof, at a gene locusin the cell can be accomplished by the co-transformation of two to fiveDNA sub-fragments, which are subjected to recombination with each otherand integration into a genetic locus in the cell having homology toportions of the sub-fragments.

The DNA construct comprising the glucoamylase gene can further include aselectable marker, thereby facilitating the selection in a host cell.For example, the selectable marker can be for transformed yeast.Examples of yeast selectable marker include markers commonly used forselecting for transformed yeast cells. Auxotrophic markers can be usedusing a gene that controls an auxotrophy, meaning that the gene enablesyeast to produce a nutrient required for the growth of the yeast.Examples genes that control auxotrophies include leucine auxotrophy(LEU2), histidine auxotrophy (HIS3), uracil auxotrophy (URA3, URA5), andtryptophan auxotrophy (TRP1).

The DNA construct may be one which is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. For example, a yeast cell may be transformed with the DNAconstruct encoding the glucoamylase, and integrating the DNA construct,in one or more copies, in the host chromosome(s). This integration isgenerally considered to be an advantage, as the DNA sequence is morelikely to be stably maintained. Integration of the DNA constructs intothe host chromosome may be performed according to conventional methods,such as by homologous or heterologous recombination.

Engineered yeast of the disclosure can include having multiple copies(two or more) of the gene encoding SEQ ID NO:1 or SEQ ID NO:4, orvariants thereof. For example, the engineered yeast can be an engineeredSaccharomyces that has at least first, second, third, and fourthexogenous nucleic acids each including a sequence encoding at least oneSEQ ID NO:1 or SEQ ID NO:4, or variants thereof. If the engineered yeastincludes multiple copies of a gene encoding the glucoamylase gene, thenucleic acid sequences of the copies can be the same or different fromone another. Exemplary methods and yeast strains that have beenengineered to include multiple copies of glucoamylase genes aredescribed in International Application serial no. PCT/US16/24249, filedMar. 25, 2016 (Miller, et al.), which is hereby incorporated byreference in its entirety.

The engineered yeast can also optionally include introduction ofexogenous nucleic acid sequences, changes to regulatory elements thateither upregulate or down regulate expression of genes; increase in genecopy numbers, and deletions or mutations that eliminate expression,reduce expression, or increase expression or activity of a gene or geneproduct. The heterologous modification can include one or more of thefollowing: the use of a promoter that is different than the nativepromoter of the desired gene; the use of a terminator that is differentthan the native terminator of the desired gene; the introduction of thegene at a location in the genome that is different than its nativelocation; the introduction of multiple copies of the desired gene.

An additional genetic modification that can be included in theengineered yeast is the alteration or introduction of an enzyme activitythat converts a low molecular weight non-glucose sugar to glucose. Forexample, one optional additional genetic modification affects orintroduces isomaltase activity in the engineered yeast during growth onglucose. Isomaltase can convert isomaltose to glucose by hydrolyzing the1,6 ether linkage in isomaltose. An isomaltase may also exhibit crossactivity for hydrolyzing the 1,4 ether linkages in maltose. The geneticmodification can cause isomaltase activity to be introduced into thecell, cause an increased amount of isomaltase in the cell, and/or causean increase in isomaltase activity.

In some embodiments further to the glucoamylase gene, the engineeredcell includes a heterologous isomaltase gene, or an isomaltase geneunder the control of a heterologous promoter that provides increasedexpression in the cell, or present in multiple copies in the cell. Forexample, an isomaltase (IMA) gene under the control of a heterologouspromoter, such as a PDC promoter can be engineered into the yeast.

Examples of isomaltase genes that can be introduced into an engineeredyeast include, but are not limited to Saccharomyces cerevisiae IMA1(P53051), Saccharomyces cerevisiae IMA2 (Q08295), Saccharomycescerevisiae IMA3 (P0CW40), Saccharomyces cerevisiae IMA4 (P0CW41),Saccharomyces cerevisiae IMA5 (P40884), Bacillus subtilis malL (O06994),Bacillus cereus malL (P21332), Bacillus coagulans malL (Q45101),Bacillus sp. malL (P29093), etc. Preferably the isomaltase gene encodesfor a polypeptide having greater than 80%, 85%, 90%, 95%, 98% or 99%sequence identity with the amino acid sequence of accession number NP011803.3 (Saccharomyces cerevisiae IMA1).

In some embodiments, the engineered yeast can further include a geneticmodification that provides a starch-degrading polypeptide that isdifferent than the glucoamylase. For example, the genetic modificationcan be one that introduces a nucleic acid encoding a differentpolysaccharide-degrading enzyme, such as an exogenous or modifiedalpha-amylase, a betaamylase, a pullulanase, or an isoamylase. Thegenetic modification may also be one that increases the amount of anendogenous or an exogenous starch-degrading polypeptide in the cell,such as by placing the gene under control of a strong promoter, orproviding the gene in multiple copies in the cell, such as multiplecopies of the gene integrated into the genome, or multiple copiespresent on a non-chromosomal construct (e.g., a plasmid).

In some embodiments, the engineered yeast can further include a geneticmodification that provides an exogenous or modified sugar transportergene (such as an isomaltose transporter); See, for example, commonlyassigned U.S. application Ser. No. 62/268,932 filed Dec. 17, 2015,entitled “Sugar Transporter-Modified Yeast Strains and Methods forBioproduct Production,” published as WO2017106739, which is herebyincorporated by reference in its entirety.

Various host cells can be transformed with a nucleic acid encoding SEQID NO:1 or SEQ ID NO:43, or a variant thereof. In some aspects, thenucleic acid including the glucoamylase gene is present in a bacterialcell. The bacterial cell can be used, for example, for propagation ofthe nucleic acid sequence or for production of quantities of thepolypeptide.

In other aspects, the host cell is a eukaryotic cell, such as a fungalcell.

In other aspects, the heterologous glucoamylase can be purified for usein an enzyme composition, either alone or in combination with otherenzymes.

In some aspects, the host cell has tolerance to a higher amount of abioderived product, such as ethanol, in the fermentation medium. In someaspects, the host cell is an “industrial yeast” which refers to anyyeasts used conventionally in ethanol fermentation. Examples includesake yeasts, shochu yeasts, wine yeasts, beer yeasts, baker's yeasts,and the like. Sake yeasts demonstrate high ethanol fermentability andhigh ethanol resistance and genetic stability. Typically, an industrialyeast has high ethanol resistance and preferably is viable at ethanolconcentrations of 10% or greater.

In exemplary aspects, the yeast including the glucoamylase gene is a S.cerevisiae yeast. Some S. cerevisiae strains have high tolerance toethanol. Various strains of ethanol tolerant yeast are commerciallyavailable, such as RED STAR™ and ETHANOL RED™ yeast (Fermentis/Lesaffre,USA), FALI™ (Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ yeast(Ethanol Technology, Wis., USA), BIOFERM™ AFT and XR (NABC-NorthAmerican Bioproducts Corporation, GA, USA), GERT STRAND (Gert Strand AB,Sweden), SUPERSTART™ (Alltech), ANGEL™ (Angel Yeast Ltd, China) andFERMIOL™ (DSM Specialties).

Industrial yeasts are typically prototrophic and therefore do not havean auxotrophic marker suitable for selecting for a transformant. If theyeast does not have the genetic background that would otherwisefacilitate retention of the glucoamylase gene of SEQ ID NO:1 or SEQ IDNO:4, or variant thereof, within the cell upon transformation, the hostcell can be engineered to introduce one or more genetic mutation(s) toestablish use of a marker gene in association with and to maintain theglucoamylase gene in the cell. For example, a commercially availableethanol tolerant yeast cell can be genetically modified prior tointroducing the glucoamylase gene in the cell.

A marker for a different auxotrophy can be provided by disrupting thegene that controls the auxotrophy. In one mode of practice, an ethanoltolerant strain of yeast is engineered to disrupt copies of one or moregenes that control auxotrophies, such as LEU2, HIS3, URA3, URA5, andTRP1. In the case of providing uracil auxotrophy, for example, a normalura3 gene of an ethanol tolerant yeast can be replaced with an ura3⁻fragment obtained from a uracil auxotrophic mutant (for example, aSaccharomyces cervisiae MT-8 strain) to disrupt the normal ura3 gene. Inthe case of a ura3 gene-disrupted strain, the presence/absence of amarker can be easily identified or selected by taking advantage of thefact that a ura3 gene-disrupted strain is able to grow in a mediumcontaining 5-fluoroorotic acid (5-FOA) while a normal ura3 strain(wild-type yeast or usual industrial yeast) is not able to grow. In thecase of a lys2 gene-disrupted strain, the presence/absence of a markercan be easily identified or selected by taking advantage of the factthat a lys2 gene-disrupted strain is able to grow in a medium containingα-aminoadipic acid while a normal lys2 strain (wild-type yeast or usualindustrial yeast) is not able to grow. Methods for disrupting anauxotrophy-controlling gene and for selectively separatingauxotrophy-controlling gene mutants may be used depending on theauxotrophy employed. Alternatively, one can employ dominant selectionmarkers, such as the amdS from Aspergillus nidulans (U.S. Pat. No.5,876,988), which allows for growth on acetamide as the sole nitrogensource; or ARO4-OFP, which allows for growth in the presence offluoro-phenylalanine (Fukuda et. al.). These markers can be usedrepeatedly using the recyclable cre-loxP system, or alternatively can beused to create auxotrophic strains that allow additional markers to beutilized.

After the host cell has been engineered to provide a desired geneticbackground for introduction of the glucoamylase gene, the gene constructis introduced into a cell to allow for expression. Methods forintroducing a gene construct into a host cell include transformation,transduction, transfection, co-transfection, electroporation. Inparticular, yeast transformation can be carried out using the lithiumacetate method, the protoplast method, and the like. The gene constructto be introduced may be incorporated into a chromosome in the form of aplasmid, or by insertion into the gene of a host, or through homologousrecombination with the gene of a host. The transformed yeast into whichthe gene construct has been introduced can be selected with a selectablemarker (for example, an auxotrophic marker as mentioned above). Furtherconfirmation can be made by measuring the activity of the expressedprotein.

The transformation of exogenous nucleic acid sequences including theglucoamylase gene can be confirmed using methods well known in the art.Such methods include, for example, nucleic acid analysis such asNorthern blots or polymerase chain reaction (PCR) amplification of mRNA,or immunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

The engineered yeast of the disclosure can be provided in any suitableform. In some aspects, the non-natural yeast is dehydrated to form a dryyeast composition. The dry yeast composition can have increased shelflife over wet compositions.

Fermentation using a host cell expressing the glucoamylase gene can beperformed in a fermentation medium made from a feedstock derived from astarch and/or sugar containing plant material, referring to a starchand/or sugar containing plant material derivable from any plant andplant part, such as tubers, roots, stems, leaves and seeds. Starchand/or sugar comprising plant material can be obtained from cereal, suchas barley, wheat, maize, rye, sorghum, millet, barley, potatoes,cassava, or rice, and any combination thereof. The starch- and/or sugarcomprising plant material can be processed, such as by methods such asmilling, malting, or partially malting. In some aspects, the starchmaterial is from corn flour, milled corn endosperm, sorghum flour,soybean flour, wheat flour, biomass derived starch, barley flour, andcombinations thereof. Starch-containing feedstocks used to form afermentation medium can be made from any of these plant materials.

In some aspects, a feedstock used to form a fermentation medium includesa treated starch. For example, the fermentation medium can include apartially hydrolyzed starch. The partially hydrolyzed starch can includehigh molecular weight dextrins and high molecular weight maltodextrins.Collectively, starch, dextrins, maltodextrins, and any other polymerizedform a glucose are glucose polymers. Partially hydrolyzed starches andpreparation thereof are well known in the art. Partially hydrolyzedstarches can be prepared by heating the starch with an acid such ashydrochloric or sulfuric acid at a high temperature and thenneutralizing the hydrolysis mixture with a suitable base such as sodiumcarbonate. Alternatively, partially hydrolyzed starches can be preparedby an enzymatic process, such as by adding alpha-amylase to a starchpreparation. An alpha amylase can cause the endohydrolysis of(1→4)-alpha-D-glucosidic linkages in polysaccharides containing three ormore (1→4)-alpha-linked D-glucose units. A partially hydrolyzed starchproduct can be used that have amounts of starch and starch degradationproducts within desired ranges. Partially hydrolyzed starch includespreparations having minimal hydrolysis (e.g., a DE of 5, having littledextrose) to preparations having substantial hydrolysis (e.g., a DE of95, predominantly dextrose).

The feedstock can be a “liquefact”, which is corn starch that hasundergone liquefaction, with a dextrose equivalents in the range ofabout 10 to about 15. A corn wet milling process can be used to providesteep-water, which can be used for fermentation. Corn kernels can besteeped and then milled, and separated into their major constituentfractions. Light steep water is a byproduct of the steeping process, andcontains a mixture of soluble proteins, amino acids, organic acids,carbohydrates, vitamins, and minerals.

In some aspects, the feedstock can be dry grind corn, i.e., most or allof the corn kernel components are included in the fermentationfeedstock. The dry-grind corn process is the most common technology forconverting corn to ethanol in the U.S. Some aspects of dry-grindprocessing differ from the wet milling process (which uses liquefact),including, but not limited to adding urea to provide sufficient nitrogenfor fermentation. The primary aspects of dry-grind processes forproducing ethanol are well known in the art.

Feedstocks derived from any of the plant materials described hereingenerally include a “glucose polymer” which refers to those polymersincluding two or more glucose residues. Shorter glucose polymersincluding glucose dimers (e.g., maltose), trimers (e.g., triose), andthose up to about 10 glucose units, which may also be referred to as“glucose oligomers.” Feedstocks of hydrolyzed starch preparations with aDE in the range of about 2 to about 20 include predominantlymaltodextrins, which include glucose polymers having a DP of 3 (540 Da)to over about 5000 (10⁶ Da). For example, a starch preparation with a DE2 includes most maltodextrins in the range of 200,000 to about 1×105 Da,and DEs in the range of about 10 to about 20 have most maltodextrins inthe range of about 540 to about 100,000 Da. Degree of polymerization(DP) refers to the number of sugar monomer residues in a glucosepolymer.

Based on the DE of the partially hydrolyzed starch, the concentrations(% wt) of glucose (DP 1), maltose (DP 2), triose (DP 3), and longerglucose polymers of DP 4+ can be known in the composition. Table 3provides concentrations (% wt) of various meric forms of glucose atvarious DE points, as understood in the art.

TABLE 3 Glucose Maltose Triose DE (DP 1) (DP 2) (DP 3) DP 4-6 DP 7+ 5 <11 2 7 90 10 <1 3 4 15 78 15 <1 6 7 21 66 20 <1 8 9 29 53 DP 4+ 28 8 8 1173 36 14 11 10 65 43 19 14 12 55 53 28 18 13 41 63 36 31 13 20 66 40 358 17 95 95 3 0.5 1.5 100 100 0 0 0

Benefits of the engineered yeast of the current disclosure allow use offermentation mediums made from feedstocks with low DEs, such asfeedstocks having a DE of less than about 40, less than about 30, lessthan about 20, less than about 15, or less than about 10. Suchfeedstocks may require the need for using exogenous starch-degradingenzymes to generate glucose for cell growth and bioproduct formation.However, starch-containing feedstocks with higher DEs can be used inmethods with engineered yeast of the disclosure, and the engineeredyeast can still provide fermentation benefits. For example, methods ofthe invention may use s feedstock including partially hydrolyzed starchhaving a DE of not greater than about 75, or not greater than about 70,and greater than about 35, or greater than about 40, and more preferablyin the range of about 45 to about 65. A DE in the range of about 45 toabout 65 means that glucose is present in the feed composition in therange of about 19% to about 40% (wt), maltose in the range of about 14to about 35% (wt), triose in the range of about 8 to about 12% (wt), andglucose polymers having a DP of 4 or greater in the range of about 17%to about 55%. The percentages are based on the total amount of all“meric” forms of glucose in the composition. Fermentation and additionof starch-containing feedstocks can be carried out to provide glucoseand glucose polymer within desired ranges as expressed as a percentageof the total amount of all “meric” forms of glucose in the composition.

In aspects of the disclosure, given production and secretion of theglucoamylase from the engineered yeast into the fermentation medium, thefermentation method may omit addition of purified or enriched commercialglucoamylase into the medium, or at least allow significantly lesscommercial glucoamylase to be used in a fermentation method. Forexample, the engineered yeast of the disclosure can allow addition ofcommercial glucoamylase to be eliminated or at least reduced by about50%, 60%, 70%, 80%, 90%, or 95%. 100% reduction can be attained usingthe yeast described herein, especially if a longer fermentation period,for example 60 hours is used. Typically, amounts of glucoamylase in therange of about 0.014-.071 AGU/g DS would be used in fermentation methodsthat do not use a glucoamylase-secreting engineered yeast.

The benefits of using yeast engineered to express a glucoamylase enzymeaccording to SEQ ID NO:1 or SEQ ID NO:4, can be understood by fermentingthe yeast in a fermentation medium made from a liquified corn mashhaving a low DE, such as not greater than about 50, not greater thanabout 35, not greater than about 30, or not greater than about 25, ornot greater than about 20, or not greater than about 15, such as in therange of about 2 to about 20, about 2 to about 15, or about 5 to about5, and fermenting the medium to generate ethanol. The feedstock used toprepare the fermentation medium may optionally be described in terms ofglucose concentration as an overall percentage of fermentablecarbohydrates (glucose and glucose polymers) in the feedstock. Forexample, the fermentation medium can be made from a starch feedstockhaving a glucose concentration of about 2% or less, or about 1% or less,such as in the range of about 0.1% to about 2%, 0.1% to about 1.5%, orto about 0.1% to about 1%. At the end of the fermentation period, theethanol concentration is 70 g/kg or greater in the fermentation media.

To determine if a yeast expressing a glucoamylase is capable ofproducing an ethanol concentration of 70 g/kg or great in thefermentation media at the end of a fermentation period, the followingtest can be conducted: First a fermentation medium using a starchfeedstock having a DE of 30+/−2 is produced by preparing a corn mash (orliquefied corn mash) using a predetermined amount of yellow dent #2 cornthat is milled and passed through a US #20 sieve. Overs (twice-groundcorn that was retained on a US #20 sieve) are added back at a X:Y ratioof overs to sieved corn (0.020 overs/total corn mass ratio). Themoisture content is measured by the halogen moisture balance method todetermine the dry weight of the milled corn. Water is added to create a32% slurry (w/w, dry weight basis). Concentrated sulfuric acid is addedto reach a pH between 5.7-5.9. Calcium chloride dihydrate powder isadded to achieve a Calcium concentration of 35 ppm. Amylase (Liquozyme™Novozymes Liquozyme Supra 2.2×) is added based on the corn dry starchweight at a dosage ratio of 2.84 kg/ton dry basis starch dosage and theslurry is transferred to a Buchi Rotovapor R-220 flask equipped with anoil bath preset at 85° C. The reaction is allowed to proceed for 2hours, stopping once the dextrose equivalents (DE) reaches 30+/−2 byreducing the temperature to between 34-36° C. The pH is adjusted to 5.0with additional concentrated sulfuric acid. The DE is determined byusing an osmometer (Advanced™ Model 3D3 and Precion system Model OsmetteXL™) Sugar and oligocarbohydrates contents are determined using HPLCwith Aminex HPX-87H column (300 mm×7.8 mm) at 60 C, 0.01N sulfuric acidmobile phase, 0.6 mL/min flow rate. To the starch feedstock a 50% ureasolution and 10% ampicillin solution are added, targeting a finalconcentration of 1900 ppm and 5 ppm, respectively. A single fermentationtypically contains 50 g of corn mash, 190 ul of 50% urea, and 2.5 ul of10% ampicillin. A typical fermentation vessel is a baffled 250 ml shakeflask fitted with an air-lock. The air lock should contain four to fivemilliliters of canola oil. The flask is inoculated to a starting OD₆₀₀of 0.1, using a cell slurry made by scraping a fresh YPD plate into 1 mlof sterile water, Inoculate the fermentation medium with an engineeredyeast that is ethanol tolerant (e.g., ETHANOL RED®) having an exogenousnucleic acid that expresses SEQ ID NO:1 or SEQ ID NO:4 to an OD₆₀₀ of0.1. Fermentation is carried out in flasks at 30° C. with shaking in anorbital shaker at 100 rpm for approximately 48 hours. At 48 hours, assample is analyzed for the concentration of ethanol, and optionallyother compounds such as glucose, by high performance liquidchromatography with refractive index detector.

Without any commercial glucoamylase supplementation and using thefeedstock described above, typical ethanol titers in the range of about115 g/kg to about 135 g/kg can be observed using an engineered yeastexpressing a glucoamylase of SEQ ID NO:1 or SEQ ID NO:4, or a variantthereof. Greater ethanol titers can be achieved with modifications tothe yeast and/or the fermentation conditions. For example, in someembodiments wherein supplemental commercial glucoamylase is added to themedium, greater amounts of ethanol can be produced, such as an amount of110 g/kg or greater, or 125 g/kg or greater, or 140 g/kg or greater, inthe fermentation medium. In addition to the higher final ethanol titers,the fermentation rate can also be increased as free glucose may nolonger be limiting the fermentation. Addition of the commercialglucoamylase further acts on starch polymers to create more glucose inthe fermentation medium, resulting in increased cell growth and higherethanol titers.

To achieve an ethanol concentration of 110 g/kg or greater in thefermentation media at the end of a fermentation period, the followingtest can be conducted. Prepare a starch feedstock and fermentation mediaas previously described. Supplement the fermentation medium withcommercial glucoamylase enzyme (Spirizyme Fuel HS, Novozymes) to providean additional glucoamylase activity in the medium. 0.097 AGU/g DS, or30% of the dose required for the wild type can provide a benefit.Glucoamylase activity (AGU) is defined as the amount of enzyme whichhydrolyzes 1 micromole maltose per minute under the standard conditions37° C., using 23.2 mM maltose in 100 mM acetate buffer pH 4.3, using areaction time of 5 minutes. Innoculate the fermentation medium with anengineered yeast that is ethanol tolerant (e.g., ETHANOL RED®) having anexogenous nucleic acid that expresses SEQ ID NO:1 or SEQ ID NO:4. Carryout fermentation for a period of 48 hours at 30° C. With the commercialglucoamylase supplementation and using a low glucose feedstock, typicalethanol titers in the range of about 110 g/kg to about 160 g/kg can beobserved.

Test 1 is a method as described in the preceding paragraphs when done at30° C. without commercial GA supplementation, Test 2 is a method asdescribed in the preceding paragraphs when done at 30° C. with 0.097AGU/g DS GA supplementation. Test 3 is a method as described in thepreceding paragraphs when done at 33.3° C. without commercial GAsupplementation. Test 4 is a method as described in the precedingparagraphs when done at 33.3° C. with 0.0.097DS GA supplementation. Apreferred yeast is one that can produce a minimum of 70 g/kg in Test 1AGU/g (all of the GA strains) and a minimum of 130 g/kg in test 4 (the2× Rmic and 4× Rde1 strains).

In further embodiments, following a period of fermentation, yeastengineered with of glucoamylases of the disclosure can provide adesirable final fermentation medium with high levels of bioproduct(e.g., ethanol) and low levels of byproduct. For example, the finalfermentation medium can have high levels of glucose (e.g., 70 g/kg orgreater, 90 g/kg or greater, 110 g/kg or greater, 125 g/kg or greater,or 140 g/kg or greater), and low levels of glucose, such as 1.0 g/kg orless (e.g., 0.9 g/kg or less, 0.8 g/kg or less, 0.7 g/kg or less, 0.6g/kg or less, or 0.5 g/kg or less). In the final fermentation mediumwith high ethanol titers, low glucose is beneficial as it improvesdownstream processes, such as separation of components (e.g., ethanol)in the final fermentation medium.

The fermentation medium includes water and preferably includesnutrients, such as a nitrogen source (such as proteins), vitamins andsalts. A buffering agent can also be present in the fermentation medium.Other components may also be present in the fermentation broth after aperiod of fermentation, such as fermentation products which canaccumulate as the fermentation progresses, and other metabolites.Optionally, the fermentation broth can be buffered with a base such ascalcium hydroxide or calcium carbonate, ammonia or ammonium hydroxide,sodium hydroxide, or potassium hydroxide in order to maintain a pH atwhich the organism functions well.

The fermentation medium can optionally include one or more of thefollowing enzymes that are different than the glucoamylase of SEQ IDNO:1 or SEQ ID NO:4, or variant thereof. Exemplary other enzymes includealpha amylases, beta-amylases, peptidases (proteases, proteinases,endopeptidases, exopeptidases), pullulanases, isoamylases, cellulases,hemicellulases, endo-glucanases and related beta-glucan hydrolyticaccessory enzymes, xylanases and xylanase accessory enzymes,acetolactate decarboxylases, cyclodextrin glycotransferases, lipases,phytases, laccases, oxidases, esterases, cutinases, granular starchhydrolyzing enzymes and other glucoamylases. These other enzymes canoptionally be added to the fermentation medium or the starch-containingfeedstock, such as by using a purified commercial preparation of theenzymes. Alternatively, one or more of the other enzymes can be secretedfrom the engineered yeast expressing SEQ ID NO:1 or SEQ ID NO:4, or froma different engineered cell.

The engineered yeast of the current disclosure can optionally bedescribed in terms of the engineered yeast's specific growth rate. Thegrowth rate of yeast can be defined by L=log(numbers) where numbers isthe number of yeast cells formed per unit volume (mL), versus T (time).

The fermentation is carried out under conditions so that fermentationcan occur. Although conditions can vary depending on the particularorganism and desired fermentation product, typical conditions include atemperature of about 20° C. or greater, and more typically in the rangeof about 30° C. or greater. During fermentation the reaction mixture canbe mixed or agitated. In some modes of practice, the mixing or agitationcan occur by the mechanical action of sparging gas to the fermentationbroth. Alternatively, direct mechanical agitation such as by an impelloror by other means can be used during fermentation.

The disclosure also provides non-natural yeast that have the ability togrow, and/or can produce a fermentation product at temperatures that aregreater than those in which yeast, such as Saccharomyces cerevisiae,typically are used in fermentation processes. For example, S. cerevisiaetypically have optimal growth at a temperature of about 30° C. However,engineered yeast of the disclosure can grow and provide excellentbioproduct (e.g., ethanol) titers at higher temperatures, and can alsoprovide low residual glucose. For example, in some embodiments using theengineered yeast of the disclosure, fermenting is carried out at atemperature in the range of 31° C. to 35° C., or 32° C. to 34° C., formost or all of a fermentation period. Even at the higher temperatures,the engineered yeast are able to generate glucoamylase activity in themedium, and promote excellent cell growth and bioproduct production.

During a fermentation process the fermentation medium can reach anelevated temperature such as about 32° C. or about 32° C. or greaterduring one or more time(s) during the fermentation process. Thetemperature can be elevated during part of the fermentation period, orduring the entire fermentation period. The temperature can be elevatedfor 5 minutes of greater, 10 minutes of greater, 30 minutes or greater,1 hour or greater, 2 hours or greater, 5 hours or greater, or 10 hoursor greater. The time of elevated temperature can also be expressed as atotal of the overall fermentation period, such as about 0.1% to 100%,about 0.1% to about 75%, about 0.1% to about 50%, about 0.1% to about25%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% toabout 2.5%, about 0.1% to about 1%, or about 0.1% to about 0.5% of thefermentation period.

The engineered yeast can also provide a commercially relevant titer ofethanol during or after the period of elevated temperature. For example,during or after the period of elevated temperature, for example, theethanol titer can be in the range of about 110 g/L to about 170 g/L, inthe range of about 125 g/L to about 170 g/L, or in the range of about140 g/L to about 170 g/L. Accordingly, the engineered yeast describedherein can produce ethanol at a commercially useful titer during orafter a period of high temperature that would typically cause issues inother currently available yeast strains used in ethanol-producingfermentation processes. Such issues include but are not limited to:death to a significant percentage of yeast cells; deleterious effects onthe ability of the yeast to reproduce; and/or reduction or eliminationof the ability of the yeast to produce a fermentation product.

Miller et al. (both WO2016127083, filed Feb. 6, 2015, andPCT/US17/045493, filed Aug. 4, 2017, which are hereby incorporated byreference in their entirety) describes the utility of swapping theleader sequence on several glucoamylases, but also highlights the needfor additional modifications to the host to achieve acceptableethanol/glucose titers at the elevated temperatures. Specifically,Miller describes the effect of expressing the Mfalpha2-R. oryzae GA onethanol and residual glucose titers at two different temperatures, 30°C. and 33.3° C. Temperature is a well-known antagonist to healthyethanol fermentations, and producers spend a significant amount ofcapital and operating cost in terms of cooling capacity to keep theirfermenters in the safe zone, typically less than 34° C. Heterologousprotein production is also a well described stressor in engineeredorganisms, as energy directed towards cell growth and maintenance isdiverted to non-natural production processes as described in Mattanovichet. al (Journal of Biotechnology 113, 2004). Alleviating the burden ofheterologous protein production has been an area of intense focus overthe past several decades, in all aspects of biotechnology (e.g. pharma,industrial enzymes, etc), and is not limited to the yeast Saccharomycescerevisiae. Classical techniques and targeted pathway engineering, twoprimary methods to overcome the obstacles of producing protein andmaintaining healthy host performance have resulted in some success(Payne et. al 2008, Gasser et. al 2007, Valkonen et. al 2003). Theseresults also indicate that there is no one solution to the problem, anda solution for one protein may not work for another.

In some cases, fermentation is carried out in industrial capacityfermenters in order to achieve commercial scale economic benefits andcontrol. In an aspect, the fermentation is carried out in a fermenterthat has a capacity of about 10,000 liters or more.

The pH of the fermentation medium can be adjusted to provide optimalconditions for glucoamylase activity, cell growth, and fermentationactivity to provide a desired product, such as ethanol. For example, pHof the solution can be adjusted to in the range of 3 to 5.5. In one modeof practice, the pH of the fermentation medium is in the range of 4 to4.5.

As noted above, the present fermentation process using geneticallymodified yeast expressing SEQ ID NO:1 or SEQ ID NO:4, or a variantthereof, and capable of secreting the enzyme produced into thefermentation medium. These enzymes are therefore directly exposed to thebroth conditions and affect the carbohydrate composition in thefermentation medium. In the fermentation medium the glucoamylase cancause hydrolysis and release of D-glucose from the non-reducing ends ofthe starch or related oligo- and polysaccharide molecules by cleavingalpha(1,4) and alpha-(1,6) glucosidic bonds.

Starch may also be acted on by one or more other amylases (e.g.,alpha-amylase) present in the fermentation medium. For example, ifalpha-amylase is present in the fermentation medium it can cause partialhydrolysis of precursor starch and cause a partial breakdown of thestarch molecules by hydrolyzing internal alpha-(1,4)-linkages.

In some modes of practice, the fermentation is carried out as a singlebatch until completion. In other modes of practice, the fermentation iscarried out as a fed batch fermentation process. In this mode ofpractice, a first portion of a total amount of starch material to befermented is added to the fermentation medium wherein the glucoamylaseenzyme acts on the starch to cause formation of glucose to be used as asubstrate for fermentation. Additional starch material can be added inone or more portions to provide more substrate for the glucoamylaseenzyme in the medium. The addition of starch can be regulated and theformation of glucose can be monitored to provide efficient fermentation.

In some modes of practice, the fermentation is carried out in acontinuous mode of operation. In this mode, multiple fermenters operatein series in which a starch hydrolysate is supplied in the firstfermenter, which is fed to second fermenter and so on until the starchhydrolysate is converted to ethanol. Continuous operation can beoperated using between 2-7 fermenters.

In some modes of practice, a portion of the total amount of starchmaterial is added to the fermentation broth using a variable rateaddition system. Examples of such systems include a variable speed pumpor a metering valve (such as a throttle valve) operably connected to apump, which pump or valve can be utilized to vary the amount of starchmaterial introduced into the fermentation broth over time. In some modesof practice, during the addition of a portion of the starch material,glucose concentration is monitored by a real-time monitoring system.

Real-time monitoring systems include systems that directly monitorglucose concentration and systems that indirectly monitor glucoseconcentration. Examples of real-time monitoring systems that typicallydirectly monitor glucose concentration include systems based on infrared(IR) spectroscopy, near-infrared (NIR) spectroscopy systems, Fouriertransform infrared (FTIR) systems, systems based on refractive index,automated enzyme based measurement systems such as a YSI 2950Biochemistry Analyzer sold by YSI Life Sciences systems, highperformance liquid chromatography (HPLC) based systems, gaschromatography (GC) based systems, and other real-time monitoringsystems known to one of skill in the art. Additionally real-timemonitoring systems that indirectly monitor/measure the glucoseconcentration of a fermentation process can be developed by determiningthe typical carbon distribution in a particular fermentation process andcorrelating the glucose concentration present in the fermentation brothto another parameter exhibited by the fermentation, such as, forexample, a correlation of the glucose level present in the fermentationbroth with a measurement of the carbon dioxide evolution rate and theamount of carbon dioxide present in an off-gas stream from thefermentation vessel. The carbon dioxide can be readily measured throughuse of a mass spectrometer or other suitable instrumental technique formeasuring the components of the off-gas stream. In a preferred aspect,the glucose concentration is monitored by a real-time monitoring systemusing infrared spectroscopy. In another one aspect, the glucoseconcentration is monitored by a real-time monitoring system usingnear-infrared spectroscopy. The real time monitoring systems interfacewith equipment that controls the introduction of starch material intothe fermentation broth to modulate the formation of glucose to a desiredconcentration in the fermentation broth.

During the fermentation process a sample of the fermentation medium canbe taken to determine the amount of glucoamylase activity in the medium.The amount of glucoamylase activity in the medium can be referred to asextracellular glucoamylase activity as it corresponds to glucoamylasesecreted from the engineered yeast. In some modes of measuring, theamount of glucoamylase activity in the medium can be determined by theamount of glucoamylase activity per amount of biomass per volume ofmedium.

Measuring the glucoamylase activity in the fermentation medium can beanother way of reflecting the benefits of using yeast engineered toexpress a glucoamylase enzyme according to SEQ ID NO:1 or SEQ ID NO:4.Such a test can be carried out by using a fermentation medium made froma low DE feedstock, high DE feedstock, or anything in between. Duringthe fermentation process a sample of medium is taken and the biomassamount and the enzyme activity are determined. As used herein “biomass”refers to the weight of the engineered yeast, which can be measured ingrams of dried cell weight per liter of medium (DCW/L).

In some modes of practice, the fermentation period is about 30 hours orgreater, about 40 hours or greater, about 50 hours or greater, or about60 hours or greater, such as a period of time in the range of about 40to about 120 hours, or 50 to about 110 hours.

The fermentation product (also referred to herein as a “bio-derivedproduct” or “bioproduct”) can be any product that can be prepared byenzymatic degradation of a starch material by the glucoamylase,formation of glucose, and fermentation of glucose. In some aspects, thefermentation product is selected from the group consisting of: aminoacids, organic acids, alcohols, diols, polyols, fatty acids, fatty acidalkyl esters (such as fatty acid methyl or ethyl esters (for example C6to C12 fatty acid methyl esters (preferably C8 to C10 fatty acid methylesters))), monacyl glycerides, diacyl glycerides, triacyl glycerides,and mixtures thereof. Preferred fermentation products are organic acids,amino acids, fatty acid alkyl esters (such as fatty acid methyl esters(for example C8 to C12 fatty acid methyl esters (preferably C8 to C10fatty acid methyl esters))), and their salts thereof, and especiallywhere the organic acid is selected from the group consisting of hydroxylcarboxylic acids (including mono-hydroxy and dihydroxy mono-, di-, andtri-carboxylic acids), monocarboxylic acids, dicarboxylic acids, andtricarboxylic acids and mixtures thereof. Examples of fermentationproducts that are prepared by the present process are organic acids oramino acids such as lactic acid, citric acid, malonic acid, hydroxybutyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid,3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid,itaconic acid, muconic acid, methacrylic acid, acetic acid, methylhexanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyldodecanoate, ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyldecanoate, ethyl dodecanoate, and mixtures thereof and derivativesthereof and salts thereof. In a preferred aspect, a fermentation methodof the disclosure produces ethanol as the bioproduct.

The fermentation product can have an excellent ratio of bioproduct(e.g., ethanol) to residual glucose, which is beneficial as it improvesdownstream processes, such as separation of components (e.g., ethanol)in the final fermentation medium. For example, the amount of glucose inthe fermentation medium is 1.0 g/kg or less, 0.9 g/kg or less, 0.8 g/kgor less, 0.7 g/kg or less, 0.6 g/kg or less, 0.5 g/kg or less, 0.4 g/kgor less, 0.3 g/kg or less, or 0.2 g/kg or less, such as a glucose amountin the range of about 0.05 g/kg to about 1.0 g/kg, or about 0.05 g/kg toabout 0.5 g/kg. The final fermentation medium can have anethanol:glucose (wt/wt) ratio of about 70:1 (wt/wt) or greater, about100:1 (wt/wt) or greater, about 150:1 (wt/wt) or greater, about 200:1(wt/wt) or greater, about 250:1 (wt/wt) or greater, or about 300:1(wt/wt) or greater, such as in the range of about 75:1 (wt/wt) to about750:1 (wt/wt), or about 100:1 (wt/wt) to about 500:1 (wt/wt).

The fermentation product is recovered from the fermentation broth. Themanner of accomplishing this will depend on the particular product.However, in some modes of practice, the organism is separated from theliquid phase, typically via a filtration step or centrifugation step,and the product recovered via, for example, distillation, extraction,crystallization, membrane separation, osmosis, reverse osmosis, or othersuitable technique.

The present process provides the ability to make fermentation productson a production scale level with excellent yields and purity. In anaspect, the process is carried out in fermentation broth quantities ofat least 25,000 gallons. In an aspect, the batch process is carried outin to produce batches of at least 25,000 gallons of final fermentationbroth. In some aspects the process is a continuous process, performed invessels of at least 200,000 gallons.

In some aspects, a genetically modified yeast expressing SEQ ID NO:1 orSEQ ID NO:4, or a variant thereof, can be used for conversion processes,such as for the production of dextrose for fructose syrups, specialtysugars and in alcohol and other end-product (e.g., organic acid,ascorbic acid, and amino acids). Production of alcohol from thefermentation of starch substrates using glucoamylases of the disclosurecan include the production of fuel alcohol or potable alcohol.

Ethanol mass yield can be calculated by dividing the ethanolconcentration by the total glucose consumed. Since glucose can bepresent as free glucose or tied up in oligomers, one needs to accountfor both. To determine the total glucose present at the beginning andend of fermentation, a total glucose equivalents measurement isdetermined. Total glucose equivalence measurement is as follows. Glucoseis measured with HPLC using RI detection. Separation is completed with aBio Rad 87H column using a 10 mM H2SO4 mobile phase. Glucose is measuredin triplicate for each sample. An acid hydrolysis is performed intriplicate in 6% (v/v) trifluoroacetic acid at 121° C. for 15 minutes.The resulting glucose after hydrolysis is measured by the same HPLCmethod. The total glucose equivalents present in each sample is theamount of glucose measured after acid hydrolysis. The total glucoseconsumed is calculated by subtracting the total glucose equivalentspresent at the end of fermentation from the total glucose equivalentspresent at the beginning of the fermentation.

Use of the engineered yeast of the current disclosure may also providebenefits with regards to increased titers, reduced volatile organicacids (VOCs), and reduced fusel oil compounds (volatile organic acids,higher alcohols, aldehydes, ketones, fatty acids and esters).

The fermentation product may be first treated with one or more agentsvia a treatment system. The treated fermentation product can then besent to a distillation system. In the distillation system, thefermentation product can be distilled and dehydrated into ethanol. Insome aspects, the components removed from the fermentation mediuminclude water, soluble components, oil and unfermented solids. Some ofthese components can be used for other purposes, such as for an animalfeed product. Other co-products, for example, syrup can be recoveredfrom the stillage.

The present disclosure also provides a method for the production of afood, feed, or beverage product, such as an alcoholic or non-alcoholicbeverage, such as a cereal- or malt-based beverage like beer or whiskey,such as wine, cider, vinegar, rice wine, soya sauce, or juice, saidmethod comprising the step of treating a starch and/or sugar containingplant material with a composition as described herein. In anotheraspect, the invention also relates to a kit comprising a glucoamylase ofthe current disclosure, or a composition as contemplated herein; andinstructions for use of said glucoamylase or composition. The inventionalso relates to a fermented beverage produced by a method using theglucoamylase.

After the fermentation process is complete, materials present in thefermentation medium can be of use. In some aspects, after a fermentationprocess has been completed, or while a fermentation process is ongoing,some or all of a bioproduct can be removed from the fermentation mediumto provide a refined composition comprising non-bioproduct solids. Thenon-bioproduct solids can include the non-natural yeast, feedstockmaterial in the medium that is not utilized by the yeast, as well asfermentation co-products. These materials can provide sources ofcarbohydrates and proteins that are useful as supplements to improve thenutritional content of a feed composition. The feed material can be aco-product from a fermentation process such as stillage (whole stillage,thin stillage, etc.) or composition prepared therefrom including drieddistillers grains (DDG), distillers dry grains with solubles (DDGS),distillers wet grains (DWG), and distillers solubles (DS).

A fermentation medium, optionally with some or all of the targetbioproduct removed, can be further treated, such as to remove water, orto cause precipitation or isolation of the non-bioproduct solids fromthe medium. In some cases the medium is treated by freeze drying or ovendrying. After treatment the refined composition may be in the form of,for example, a liquid concentrate, a semi-wet cake, or a dry solid. Therefined composition can be used as a feed composition itself, or aningredient in the preparation of a feed composition. In preferredpreparations, the feed composition is a livestock feed composition suchas for sheep, cattle, pigs, etc.

The solids in the fermentation medium can provide a source of one ormore amino acids. Introduced into an animal feed, the fermentationco-product can provide an enhanced amino acid content with regard to oneor more essential amino acids. Essential amino acids can includehistidine, isoleucine, lysine, methionine, phenylalanine, threonine, andtryptophan. These amino acids can be present in the feed composition asfree amino acids or can be derived from proteins or peptides rich in theamino acids. The solids in the fermentation medium can provide a sourceof one or more prebiotics, which are nondigestible food substances, suchas nondigestible oligosaccharides, that selectively stimulate the growthof favorable species of bacteria in the gut, thereby benefitting thehost. The solids in the fermentation medium can provide a source ofphytases, β-glucanases, proteases, and xylanases.

Table 4 includes strains used in the experimental studies associatedwith the disclosure.

TABLE 4 Strain ID Strain Description Strain 1 Wild Type Strain 1-1 Ura3ΔStrain 1-2 2X Mfa2-R. oryzae GA Strain 1-3 4X Mfa-R. oryzae GA Strain1-4 1X R. microsporus GA Strain 1-5 2X R. microsporus GA Strain 1-6 2XR. microsporus GA Strain 1-7 4X R. microsporus GA Strain 1-8 2X R.delemar GA Strain 1-9 4X R. delemar GA

Example 1 Screening a Diverse Library of Glucoamylase Enzymes for Growthon Starch

Heterologous expression of a functional glucoamylase in Saccharomycescerevisiae was first demonstrated circa 1993, using the Aspergillusniger glucoamylase. Other uses of glucoamylase in Saccharomycescerevisiae have been reported, but still represent only a very smallfraction of the number of public sequence information for theseproteins. To that aim, over 1,000 enzymes were expressed and tested froma diverse set of organisms to identify enzymes that confer the desiredtrait of high glucoamylase expression while maintaining ethanol rate,titer, and yield.

A DNA library was constructed containing 1037 genes encodingglucoamylases, alpha-amylases, amylopullulanases, or other starchhydrolyzing enzymes by cloning synthetically created open reading framesinto a Saccharomyces cerevisiae episomal plasmid. The enzymes encoded bythese genes were sourced from four distinct classes including: 1)enzymes that were annotated with a glucoamylase EC number (whichincluded both glucoamylases and a-amylases), 2) enzymes that wereannotated as having both α-1,6 and α-1,4 glycosidase activity 3)structural homologs of previously identified functional fungalglucoamylases expressed in Saccharomyces cerevisiae, and finally 4)starch hydrolyzing enzymes from ruminant gut microbiomes. Each enzyme inthe library was screened with its native leader as well as onesubstituted with the Saccharomyces cerevisiae Mfα2 leader. In total,1,773 plasmids were successfully transformed into Strain 1-1 (describedin previous application). Resulting transformants were tested for growthon starch containing media, using iodine staining to reveal zones ofclearing. A total of 245 strains were able to produce zones of clearing,indicating that they contained plasmids with genes encoding heterologousenzymes capable of generating starch hydrolyzing activity when expressedin a yeast. These 245 were further screened for ethanol production asdescribed below. The remaining genes encoded by the remaining 1528plasmids were deemed not to be sufficiently active to warrant furthertesting.

Secondary Screening for Ethanol Production in Deep Well Microtiters

Ethanol production was measured using deep well microtiter platescontaining 0.5 mL of media. The fermentation medium consists of 725 gpartially hydrolyzed corn starch in the form of liquifact, 150 gfiltered light steep water, 125 g sterile water, 25 g glucose, and 1 gurea. Partially hydrolyzed corn starch is provided by Cargill'sEddyville, Iowa corn wet mill (DS 30-37%, DE 5-15). Light steep water isalso provided from Cargill's Eddyville, Iowa corn wet mill (freeavailable nitrogen 2000-2500 ppm). Light steep water is centrifuged at8,000 RPM, and the resulting supernatant is filter sterilized using 0.2micron filters to produce filtered light steep water. Strains areinoculated to an OD₆₀₀ of 0.1 and the plate is incubated at 30° C. withshaking in an orbital shake at 1000 rpm. Samples are taken and analyzedfor relevant metabolite concentrations at the end of fermentation byHPLC.

Selected results from the screening of the 245 strains is shown in Table5. Most of the strains did not demonstrate commercially relevant ethanoltiters (e.g., Aspergillus kawachii, Aspergillus terreus, Thermomyceslanuginosus, and Mfα2-Neurospora crassa are representative of suchstrains). However, two strains (Rhizopus delemar and Rhizopusmicrospores) demonstrated ethanol titers greater than commerciallyrelevant strains known in the art (Mfα2-Rhizopus oryzae andSaccharomycopsis fibuligera).

TABLE 5 Ethanol titer (g/L) at Enzyme Source, Accession # SEQ ID NO61.25 hours Rhizopus delemar, I1BGP8 SEQ ID NO 7 100.08 Rhizopusmicrosporus, SEQ ID NO 8 107.82 A0A0C7BD37 Mfα2-Rhizopus oryzae, Q2VC81SEQ ID NO 9 99.50 Saccharomycopsis fibuligera, SEQ ID NO 10 89.64 Q8TFE5Aspergillus kawachii, G7XVA6 SEQ ID NO 11 33.70 Aspergillus terreus,Q0CPK9 SEQ ID NO 12 34.20 Thermomyces lanuginosus, SEQ ID NO 13 35.00Q58HN1 Mfα2-Neurospora crassa, SEQ ID NO 14 37.90 A0A0B0E9D9

Example 2

Construction of Strains Expressing the MFalpha2-R. oryzae GA.

Creation of a ura3Δ auxotrophic base strain is previously described in(CAR0233P1 Strain 1-3), referred to as Strain 1-1 herein. Strain 1-1 istransformed with SEQ ID NO: 15 and SEQ ID NO: 16. SEQ ID NO: 15 containsthe following elements: homology to integration locus A (3986 bp), aScTDH3 promoter (992-1673 bp), a Rhizopus oryzae glucoamylase withmodified signal sequence (1680-3476 bp), a ScCYC1 terminator (3485-3708bp), a loxP recombination site (3717-3750 bp), a ScURA3 promoter(3751-4257 bp), the upstream portion of the ScURA3 (4258-4861 bp). SEQID NO: 16 contains the following elements: downstream portion of theScURA3 (7-606 bp), a ScURA3 terminator (607-927 bp), a loxPrecombination site (928-961 bp), a ScPGK1 promoter (968-1554 bp), aRhizopus oryzae glucoamylase with modified signal sequence (1561-3357bp), a ScGAL10 terminator (3366-3836 bp), and homology to integrationlocus A (3838-4748 bp). Transformants are selected on synthetic completemedia lacking uracil. (ScD-Ura). Resulting transformants are streakedfor single colony isolation on ScD-Ura. A single colony is selected.Correct integration of SEQ ID NO: 15 and SEQ ID NO: 16 into one alleleof integration locus A is verified by PCR in the single colony. A PCRverified isolate is designated Strain 1-2 (yNS220).

Strain 1-2 is transformed with SEQ ID NO: 17, 18 and 19. SEQ ID NO: 17contains the following elements: homology to integration locus A (3-986bp), a ScTDH3 promoter (9921673 bp). SEQ ID NO: 18 contains thefollowing elements: a ScTDH3 promoter (6-687 bp), a Rhizopus oryzaeglucoamylase with modified signal sequence (694-2490), a ScCYC1terminator (2499-2722 bp), a loxP recombination site (2731-2674 bp), aScTEF1 promoter (2765-3220 bp), and the upstream portion of theAspergillus nidulans acetamidase (3221-4260). SEQ ID NO: 19 contains thefollowing elements: the downstream portion of the Aspergillus nidulansacetamidase (7-1032 bp), a ScADH1 terminator (1033-1335 bp), a loxPrecombination site (1336-1369 bp), a ScPGK1 promoter (1376-1962 bp), aRhizopus oryzae glucoamylase with modified signal sequence (1969-3765bp), a ScGAL10 terminator (3774-4244 bp), and homology to integrationlocus A (4246-5008 bp). Transformants are selected on Yeast NitrogenBase (without ammonium sulfate or amino acids) containing 20 g/L glucoseand 1 g/L acetamide as the sole nitrogen source. Resulting transformantsare streaked for single colony isolation on Yeast Nitrogen Base (withoutammonium sulfate or amino acids) containing 20 g/l glucose and 1 g/Lacetamide as the sole nitrogen source. A single colony is selected.Correct integration of SEQ ID NO 17, 18 and 19 into the second allele oflocus A is verified by PCR in the single colony. A PCR verified isolateis designated Strain 1-3.

Example 3

Construction of Strains Expressing the Rhizopus microsporus GA.

Strain 1-1 is transformed with SEQ ID NO: 20 and SEQ ID NO: 21. SEQ IDNO: 20 contains the following elements: homology to integration locus A(3-986 bp), a ScTDH3 promoter (992-1673 bp), a Rhizopus microsporusglucoamylase (1680-3497 bp), a ScCYC1 terminator (3506-3729 bp), a loxPrecombination site (3738-377 lbp), a ScURA3 promoter (3772-4278 bp), theupstream portion of the ScURA3 (4279-4882 bp). SEQ ID NO: 21 containsthe following elements: A portion of the ScURA3 promoter (11-446), aScURA3 (447-1250 bp), a ScURA3 terminator (1251-1570 bp), a loxPrecombination site (1571-1604 bp), and homology to integration locus A(1613-1790 bp). Transformants are selected on synthetic complete medialacking uracil. (ScD-Ura). Resulting transformants are streaked forsingle colony isolation on ScD-Ura. A single colony is selected. Correctintegration of SEQ ID NO: 20 and SEQ ID NO: 21 into one allele ofintegration locus A is verified by PCR in the single colony. A PCRverified isolate is designated Strain 1-4.

Strain 1-4 is transformed with SEQ ID NO: 22 and SEQ ID NO: 23. SEQ IDNO: 22 contains the following elements: homology to integration locus A(1-193 bp), a ScTDH3 promoter (199-880 bp), a Rhizopus microsporusglucoamylase (887-2704 bp), a ScCYC1 terminator (2713-2936 bp), a loxPrecombination site (2945-2978 bp), a ScTEF1 promoter (2979-3434 bp), andthe upstream portion of the Aspergillus nidulans acetamidase (3435-4474bp). SEQ ID NO 23 contains the following elements: the downstreamportion of the Aspergillus nidulans acetamidase (1-1498 bp), a ScTEF1terminator (1499-1658 bp), a loxP recombination site (1692-1659 bp), andhomology to integration locus A (1701-1878). Transformants are selectedon Yeast Nitrogen Base (without ammonium sulfate or amino acids)containing 20 g/L glucose and 1 g/L acetamide as the sole nitrogensource. Resulting transformants are streaked for single colony isolationon Yeast Nitrogen Base (without ammonium sulfate or amino acids)containing 20 g/l glucose and 1 g/L acetamide as the sole nitrogensource. A single colony is selected. Correct integration of SEQ ID NO:22 and SEQ ID NO: 23 into the second allele of locus A is verified byPCR in the single colony. A PCR verified isolate is designated Strain1-5.

Strain 1-1 is transformed with SEQ ID NO: 20 and SEQ ID NO: 24. SEQ IDNO: 24 contains the following elements: downstream portion of the ScURA3(7-606 bp), a ScURA3 terminator (607-927 bp), a loxP recombination site(928-961 bp), a ScPGK1 promoter (9681554 bp), a Rhizopus microsporusglucoamylase (1561-3378 bp), a ScGAL10 terminator (3387-3857 bp), andhomology to integration locus A (3859-4823 bp). Transformants areselected on synthetic complete media lacking uracil. (ScD-Ura).Resulting transformants are streaked for single colony isolation onScD-Ura. A single colony is selected. Correct integration of SEQ ID NO:20 and SEQ ID NO: 24 into one allele of integration locus A is verifiedby PCR in the single colony. A PCR verified isolate is designated Strain1-6.

Strain 1-6 is transformed with SEQ ID NO: 22 and SEQ ID NO: 25. SEQ IDNO: 25 contains the following elements: the downstream portion of theAspergillus nidulans acetamidase (7-1032 bp), a ScADH1 terminator(1033-1335 bp), a loxP recombination site (1336-1369 bp), a ScPGK1promoter (1376-1962 bp), a Rhizopus microsporus glucoamylase (1969-3786bp), a ScGAL10 terminator (3795-4265 bp), and homology to integrationlocus A (4267-4684 bp). Transformants are selected on Yeast NitrogenBase (without ammonium sulfate or amino acids) containing 20 g/L glucoseand 1 g/L acetamide as the sole nitrogen source. Resulting transformantsare streaked for single colony isolation on Yeast Nitrogen Base (withoutammonium sulfate or amino acids) containing 20 g/l glucose and 1 g/Lacetamide as the sole nitrogen source. A single colony is selected.Correct integration of SEQ ID NO: 22 and SEQ ID NO: 25 into the secondallele of locus A is verified by PCR in the single colony. A PCRverified isolate is designated Strain 1-7.

Example 4

Construction of Strains Expressing the Rhizopus delemar GA.

Strain 1-1 is transformed with SEQ ID NO: 26 and SEQ ID NO: 27. SEQ IDNO: 26 contains the following elements: homology to integration locus A(3-986 bp), a ScTDH3 promoter (992-1673 bp), a Rhizopus delemarglucoamylase (1698-3494 bp), a ScCYC1 terminator (3503-3726 bp), a loxPrecombination site (3735-3768 bp), a ScURA3 promoter (3769-4275 bp), theupstream portion of the ScURA3 (4276-4879 bp). SEQ ID NO: 27 containsthe following elements: downstream portion of the ScURA3 (7-606 bp), aScURA3 terminator (607-927 bp), a loxP recombination site (928-961 bp),a ScPGK1 promoter (968-1554 bp), a Rhizopus delemar glucoamylase(1561-3375 bp), a ScGAL10 terminator (3384-3854 bp), and homology tointegration locus A (3856-4820). Transformants are selected on syntheticcomplete media lacking uracil. (ScD-Ura). Resulting transformants arestreaked for single colony isolation on ScDUra. A single colony isselected. Correct integration of SEQ ID NO: 26 and SEQ ID NO: 27 intoone allele of integration locus A is verified by PCR in the singlecolony. A PCR verified isolate is designated Strain 1-8.

Strain 1-8 is transformed with SEQ ID NO: 28 and SEQ ID NO: 29. SEQ IDNO: 28 contains the following elements: homology to integration locus A(1-986 bp), a ScTDH3 promoter (992-1673 bp), a Rhizopus delemarglucoamylase (1680-3494 bp), a ScCYC1 terminator (3503-3726 bp), a loxPrecombination site (3735-3768 bp), a ScTEF1 promoter (3769-4224 bp), andthe upstream portion of the Aspergillus nidulans acetamidase (4225-5264bp). SEQ ID NO: 29 contains the following elements: the downstreamportion of the Aspergillus nidulans acetamidase (7-1032 bp), a ScADH1terminator (1033-1335 bp), a loxP recombination site (1336-1369 bp), aScPGK1 promoter (1376-1962 bp), a Rhizopus delemar glucoamylase(1969-3783 bp), a ScGAL10 terminator (3792-4262 bp), and homology tointegration locus A (4264-5026 bp). Transformants are selected on YeastNitrogen Base (without ammonium sulfate or amino acids) containing 20g/L glucose and 1 g/L acetamide as the sole nitrogen source. Resultingtransformants are streaked for single colony isolation on Yeast NitrogenBase (without ammonium sulfate or amino acids) containing 20 g/l glucoseand 1 g/L acetamide as the sole nitrogen source. A single colony isselected. Correct integration of SEQ ID NO: 28 and SEQ ID NO: 29 intothe second allele of locus A is verified by PCR in the single colony. APCR verified isolate is designated Strain 1-9.

Example 5 Characterization of Strains in 32% DS Corn Mash at 30° C.(TEST #1 and #2).

Strain 1, 1-3, 1-5, 1-7, 1-9 are struck to a YPD plate and incubated at30° C. until single colonies are visible (1-2 days). Cells from the YPDplate are scraped into pH 7.0 phosphate buffer and the optical density(OD600) is measured. Optical density is measured at wavelength of 600 nmwith a 1 cm path length using a model Genesys20 spectrophotometer(Thermo Scientific). A shake flask is inoculated with the cell slurry toreach an initial OD600 of 0.1. Immediately prior to inoculating thefollowing materials are added to each flask: 50 grams of liquified cornmash (32% DS, DE 30+/−2) is added to a 250 mL baffled shake flask sealedwith air-lock containing 4 mls of sterilized canola oil, 190 ul of 500g/L filter-sterilized urea, and 2.5 ul of 100 mg/ml of filter sterilizedampicillin.

0.324 AGU/g DS (70 μl of a 1:10 dilution) of glucoamylase (SpirizymeFuel HS, Novozymes) is added to flasks containing the control Strain 1,and either zero or 0.097 AGU/g DS (21 μL of a 1:10 dilution ofglucoamylase (Spirizyme Fuel HS™, Novozymes is added to the remainingflasks depending on the “Test”. Spirizyme Fuel HS™ is estimated to haveapproximately 769 AGU/g enzyme, however over time the activity canchange 10-20% (i.e., the activity of the enzyme typically decreases overtime). Duplicate flasks for each strain are incubated at 30° C. withshaking in an orbital shaker at 100 rpm for approximately 48 hours. At48 hours, 1 ml samples are taken and analyzed for ethanol and glucoseconcentrations in the broth by high performance liquid chromatographywith refractive index detector. Selected results are shown in Table 6.

TABLE 6 Test #1 Test #1 Final EtOH titer Residual Glucose (g/kg) (g/kg)Strain 1, with 0.324 AGU/g DS 133.6 +/− 1.6 0.4 +/− 0.1 Strain 1-3, nosupplementation 132.5 +/− 0.3 0.2 +/− 0.0 Strain 1-5, no supplementation120.9 +/− 1.3 0.5 +/− 0.1 Strain 1-7, no supplementation 134.1 +/− 4.80.2 +/− 0.0 Strain 1-9, no supplementation 119.8 +/− 1.9 0.9 +/− 0.0Test #2 Test #2 Final EtOH titer Residual Glucose (g/kg) (g/kg) Strain1, with 0.324 AGU/g DS 133.6 +/− 1.6 0.4 +/− 0.1 Strain 1-3, nosupplementation 130.7 +/− 2.7 0.2 +/− 0.1 Strain 1-5, no supplementation132.9 +/− 0.2 0.3 +/− 0.0 Strain 1-7, no supplementation 135.0 +/− 1.20.3 +/− 0.0 Strain 1-9, no supplementation 131.9 +/− 0.5 0.2 +/− 0.1

Example 6 Characterization of Strains in 32% DS Corn Mash at 33.3° C.(TEST #3 and #4).

Strain 1, 1-3, 1-5, 1-7, 1-9 are struck to a YPD plate and incubated at30° C. until single colonies are visible (1-2 days). Cells from the YPDplate are scraped into pH 7.0 phosphate buffer and the optical density(OD₆₀₀) is measured. Optical density is measured at wavelength of 600 nmwith a 1 cm path length using a model Genesys20 spectrophotometer(Thermo Scientific). A shake flask is inoculated with the cell slurry toreach an initial OD₆₀₀ of 0.1. Immediately prior to inoculating thefollowing materials are added to each flask: 50 grams of liquified cornmash is added to a 250 mL baffled shake flask sealed with air-lockcontaining 4 mls of sterilized canola oil, 190 ul of 500 g/Lfilter-sterilized urea, and 2.5 ul of 100 mg/ml of filter sterilizedampicillin 0.324 AGU/g DS (70 μl of a 1:10 dilution) of glucoamylase(Spirizyme Fuel HS™, Novozymes) is added to flasks containing thecontrol Strain 1, and either zero or 0.097 AGU/g DS (21 μl of a 1:10dilution) of glucoamylase (Spirizyme Fuel HS™, Novozymes) is added tothe remaining flasks, depending on the “Test”. Spirizyme Fuel HS™ isestimated to have approximately 326 AGU/g enzyme. Duplicate flasks foreach strain are incubated at 33.3° C. with shaking in an orbital shakeat 100 rpm for approximately 48 hours. At 48 hours, 1 ml samples aretaken and analyzed for ethanol and glucose concentrations in the brothby high performance liquid chromatography with refractive indexdetector. Selected results are shown in Table 7.

TABLE 7 Test #3 Test #3 Final EtOH titer Residual Glucose (g/kg) (g/kg)Strain 1, with 0.324 AGU/g DS 135.8 +/− 0.4 0.8 +/− 0.1 Strain 1-3, nosupplementation 124.1 +/− 4.4 6.0 +/− 0.0 Strain 1-5, no supplementation132.8 +/− 1.2 0.2 +/− 0.0 Strain 1-7, no supplementation 131.2 +/− 2.42.6 +/− 0.2 Strain 1-9, no supplementation 132.9 +/− 1.2 0.2 +/− 0.0Test #4 Test #4 Final EtOH titer Residual Glucose (g/kg) (g/kg) Strain1, with 0.324 AGU/g DS 135.8 +/− 0.4 0.8 +/− 0.1 Strain 1-3 with 0.097AGU/g 129.5 +/− 3.6 3.3 +/− 0.0 DS Strain 1-5, with 0.097 AGU/g 132.8+/− 0.3 0.4 +/− 0.2 DS Strain 1-7, with 0.097 AGU/g 129.5 +/− 0.9 7.9+/− 0.2 DS Strain 1-9, with 0.097 AGU/g 134.5 +/− 1.5 0.9 +/− 0.1 DS

Example 7 Characterization of Strains in 34% DS Corn Mash at 33.3° C.

Strains 1, 1-3, 1-6, and 1-9 are struck to a YPD plate and incubated at30° C. until single colonies are visible (1-2 days). Cells from the YPDplate are scraped into pH 7.0 phosphate buffer and the optical density(OD₆₀₀) is measured. Optical density is measured at wavelength of 600 nmwith a 1 cm path length using a model Genesys20 spectrophotometer(Thermo Scientific). A shake flask is inoculated with the cell slurry toreach an initial OD₆₀₀ of 0.1. Immediately prior to inoculating thefollowing materials are added to each flask: 50 grams of liquified cornmash is added to a 250 mL baffled shake flask sealed with air-lockcontaining 4 mls of sterilized canola oil, 190 ul of 500 g/Lfilter-sterilized urea, and 2.5 ul of 100 mg/ml of filter sterilizedampicillin 0.324 AGU/g DS (70 μl of a 1:10 dilution) of glucoamylase(Spirizyme Fuel HS™, Novozymes) is added to flasks containing thecontrol Strain 1, and either zero or 0.032 AGU/g DS, 0.065 AGU/g DS,0.097 AGU/g DS, or 0.162 AGU/gDS. (7 μl, 14 μl, 21 μl, or 35 μl of a1:10 dilution) is added to the remaining flasks. Spirizyme Fuel HS™ isestimated to have approximately 326 AGU/g enzyme. Duplicate flasks foreach strain are incubated at 33.3° C. with shaking in an orbital shakeat 100 rpm for approximately 48 hours. At various intervals, the flasksare opened and samples are analyzed for ethanol and glucoseconcentrations in the broth by high performance liquid chromatographywith refractive index detector. Selected results are shown in Table 8.

TABLE 8 Final EtOH titer Residual Glucose (g/kg) (g/kg) Strain 1, 0.324AGU/g DS dose 137.0 +/− 2.8 1.2 +/− 0.3 Strain 1-3, no supplementation123.2 +/− 1.1 12.1 +/− 0.5  Strain 1-3, 0.032 AGU/g DS dose 122.3 +/−3.2 14.8 +/− 0.8  Strain 1-3, 0.065 AGU/g DS dose 123.4 +/− 4.2 16.8 +/−0.0  Strain 1-3, 0.097 AGU/g dose DS 130.1 +/− 1.4 14.6 +/− 0.3  Strain1-6, no GA dose 133.9 +/− 5.1 0.2 +/− 0.1 Strain 1-6, 0.032 AGU/g doseDS 137.5 +/− 3.7 0.6 +/− 0.0 Strain 1-6, 0.097 AGU/g dose DS 138.8 +/−0.2 1.0 +/− 0.0 Strain 1-6, 0.162 AGU/gDS dose 138.4 +/− 2.8 2.0 +/− 0.7Strain 1-9, no supplementation 135.1 +/− 0.3 1.2 +/− 0.1 Strain 1-9,0.032 AGU/g DS dose 136.2 +/− 5.4 1.4 +/− 0.2 Strain 1-9, 0.097 AGU/g DSdose 135.7 +/− 1.0 4.8 +/− 0.9 Strain 1-9, 0.162 AGU/g DS dose 136.0 +/−2.0 6.1 +/− 0.3

Example 8 Characterization of a Strain in 32% DS Corn Mash Having a DEof 30+/−2 at a Temperature of 30° C.

A strain is struck to a YPD plate (20 g/L yeast peptone, 10 g/L yeastextract, 20 g/L glucose, and 20 g/L agar) and incubated at 30° C. untilsingle colonies are visible (1-2 days). Cells from the YPD plate arescraped into pH 7.0 phosphate buffer to create a cell slurry and theoptical density (OD₆₀₀) is measured. Optical density is measured atwavelength of 600 nm with a 1 cm path length using a model Genesys20spectrophotometer (Thermo Scientific). A shake flask is inoculated withthe cell slurry to reach an initial OD₆₀₀ of 0.1. Immediately prior toinoculating the following materials are added to each flask: 50 grams ofliquified corn mash (32% DS, DE 30) is added to a 250 mL baffled shakeflask sealed with air-lock containing 4 mls of sterilized canola oil,190 ul of 500 g/L filter-sterilized urea, and 2.5 ul of 100 mg/ml offilter sterilized ampicillin. The shake flask, airlock, canola oil isweighed prior to the addition of the fermentation media and cells, whichis subtracted from the total weight of the flask to give the startingmedia volume. At various time points in the fermentation, the flasks areremoved and the weight recorded. The mass loss (grams) at any timepointis calculated by subtracting the mass at T1 from the original mass atT0. The mass loss (grams) is converted to a mass loss (percentage) bydividing the mass loss at any given time point by the original startingmass. The percentage mass loss can be converted to g/kg ethanol by thefollowing equation (the starting mass of the fermentation media. Ethanol(g/kg)=(Percent mass loss+0.0016)/0.0009/1.042.

Exemplary Embodiments

A. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein the yeast is capable of producingethanol at a rate of 1 g/L*h or greater during a fermentation process.

B. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein the yeast is capable of producing (a)at least 70 g/kg of ethanol in a fermentation medium made from a glucosepolymer-containing feedstock having (i) a DE of not greater than 50.

C. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein the yeast is capable of producing (a)at least 70 g/kg of ethanol in a fermentation medium made from corn mashhaving a DE of 30+/−2, wherein the fermentation medium comprises 32% drywt corn, and a pH 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin,wherein the staring yeast concentration is 0.1 (OD600), and fermentationis carried out at 48 hrs at 30° C. with agitation.

D. The engineered yeast of any of embodiments A-C wherein theglucoamylase comprises a sequence having 85% or greater sequenceidentity to SEQ ID NO:1.

E. The engineered yeast of embodiment D wherein the glucoamylasecomprises a sequence having 90% or greater sequence identity to SEQ IDNO:1.

F. The engineered yeast of embodiment E wherein the glucoamylasecomprises a sequence having 95%, 96%, 97%, 98%, or 99%, or greatersequence identity to SEQ ID NO:1.

G. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 97% or greater sequenceidentity to SEQ ID NO:4, wherein the yeast is capable of producingethanol at a rate of 1 g/L*h or greater during a fermentation process.

H. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 97% or greater sequenceidentity to SEQ ID NO:4, wherein the yeast is capable of producing (a)at least 70 g/kg of ethanol in a fermentation medium made from a glucosepolymer-containing feedstock having (i) a DE of not greater than 50.

I. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:4, wherein the yeast is capable of producing (a)at least 70 g/kg of ethanol in a fermentation medium made from corn mashhaving a DE of 30, wherein the corn mash is present in a fermentationmedium having 32% wt corn mash, and a pH 5.8, 35 ppm CaCl, 1900 ppmurea, 5 ppm ampicillin, wherein the staring yeast concentration is 0.1(OD600), and fermentation is carried out at 48 hrs at 30° C. withagitation.

J. The engineered yeast of any of embodiments G-I wherein theglucoamylase comprises a sequence having 98% or greater sequenceidentity to SEQ ID NO:4.

K. The engineered yeast of embodiment J wherein the glucoamylasecomprises a sequence having 99% or greater sequence identity to SEQ IDNO:4.

L. The engineered yeast of any of the above embodiments wherein thereare 2-8 copies of the exogenous nucleic acid in the cell.

M. The engineered yeast of embodiment L wherein there are 2-6 copies ofthe exogenous nucleic acid in the cell.

N. The engineered yeast of embodiment M wherein there are 4 copies ofthe exogenous nucleic acid in the cell.

0. The engineered yeast of any of the above embodiments wherein theexogenous nucleic acid is under the control of a promoter selected fromthe group consisting of a phosphoglycerate kinase (PGK) promoter nucleicacid sequence, cytochrome c promoter (pCYC) nucleic acid sequence,translational elongation factor promoter (pTEF) nucleic acid sequence,glyceraldehyde-3phosphate dehydrogenase promoter (pGPD/TDH3) nucleicacid sequence, the phosphoglycerate kinase promoter (PGK) nucleic acidsequence, and the alcohol dehydrogenase promoter (pADH) nucleic acidsequence.

P. The engineered yeast of any of the above embodiments which is aspecies of Saccharomyces.

Q. The engineered yeast of embodiment P which is Saccharomycescerevisiae.

R. The engineered yeast of any of the above embodiments which istolerant to growth in fermentation medium having a concentration ofethanol of greater than 90 g/L.

S. The engineered yeast of any of the above embodiments which istolerant to growth at temperatures in the range of greater than 31°C.-35° C.

T. The engineered yeast of embodiment S which is tolerant to growth inat temperatures in the range of greater than 32° C.-34° C.

U. The engineered yeast of any one of the above embodiments thatproduces a greater amount of ethanol than a parent strain that does notinclude the exogenous nucleic acid under the same fermentationconditions.

V. A fermentation method for producing a fermentation product,comprising a step of: forming a fermentation medium from a glucosepolymer-containing feedstock; and fermenting the fermentation mediumusing an engineered yeast comprising an exogenous nucleic acid encodinga glucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein fermenting produces the bioproduct.

W. A fermentation method for producing a fermentation product,comprising a step of: forming a fermentation medium from a glucosepolymer-containing feedstock; and fermenting the fermentation mediumusing an engineered yeast comprising an exogenous nucleic acid encodinga glucoamylase comprising a sequence having 97% or greater sequenceidentity to SEQ ID NO:4, wherein fermenting produces the bioproduct.

X. The fermentation method of embodiments V or W wherein the glucosepolymer-containing feedstock or the fermentation medium, at thebeginning of fermentation, has a DE of about 50 or less.

Y. The fermentation method of embodiments V or W wherein thefermentation medium, at the beginning of fermentation, has a glucoseconcentration of about 30 g/kg or less.

Z. The fermentation method of embodiments V or W wherein glucosepolymer-containing feedstock comprises glucose polymer having a degreeof polymerization of 4 or greater and present in an amount of 75% weightor greater total fermentable carbohydrates in the feedstock.

AA. The fermentation method of any of embodiments V-Z wherein glucosepolymer-containing feedstock is obtained from corn.

BB. The fermentation method of embodiment AA wherein glucosepolymer-containing feedstock is obtained from corn mash.

CC. The fermentation method of any of embodiments V-BB whereinfermenting is carried out for a fermentation time of at least 30 hours.

DD. The fermentation method of embodiment CC wherein fermenting iscarried out for a fermentation time in the range of 30-100 hours.

EE. The fermentation method of embodiment DD wherein fermenting iscarried out for a fermentation time in the range of 40-60 hours.

FF. The fermentation method of any of embodiments V-EE wherein saidfermenting is carried out at a temperature in the range of 31° C. to 35°C. for most or all of a fermentation period.

GG. The fermentation method of embodiment FF wherein the fermenting iscarried out at a temperature in the range of 32° C. to 34° C. for mostor all of the fermentation period.

HH. The fermentation of any of embodiments V-GG wherein ethanol isproduced to a concentration of 70 g/L or greater in the medium.

II. The fermentation of embodiment HH wherein ethanol is produced to aconcentration of 90 g/L or greater in the medium.

JJ. The method of embodiment II wherein said fermenting provides ethanolin the range of 90 g/L to 150 g/L.

KK. The method of embodiment JJ wherein said fermenting provides ethanolin the range of 110 g/L to 150 g/L.

LL. The fermentation method of any of embodiments V-KK wherein thefermentation medium has an amount of glucose of not greater than 1.0 g/Lat the end of the fermentation period.

MM. The fermentation method of embodiment LL wherein the fermentationmedium has an amount of glucose of not greater than 0.8 g/L at the endof the fermentation period.

NN. The fermentation method of any of embodiments V-MM comprising addingsupplemental glucoamylase to the feedstock, or supplemental glucoamylaseto the medium during the fermentation period.

OO. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein the yeast is capable of producing atleast 70 g/kg of ethanol in the fermentation process of Example 8.

PP. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 97% or greater sequenceidentity to SEQ ID NO:4, wherein the yeast is capable of producing atleast 70 g/kg of ethanol in the fermentation process of Example 8.

1. An engineered yeast comprising an exogenous nucleic acid encoding aglucoamylase comprising a sequence having 81% or greater sequenceidentity to SEQ ID NO:1, wherein the yeast is capable of producingethanol at a rate of 1 g/L*h or greater during a fermentation process.2. The engineered yeast of claim 1, wherein the yeast is capable ofproducing (a) at least 70 g/kg of ethanol in a fermentation medium madefrom a glucose polymer-containing feedstock having (i) a DE of notgreater than
 50. 3. The engineered yeast of claim 1, wherein the yeastis capable of producing (a) at least 70 g/kg of ethanol in afermentation medium made from corn mash having a DE of 30+/−2, whereinthe fermentation medium comprises 32% dry wt corn, and a pH 5.8, 35 ppmCaCl, 1900 ppm urea, 5 ppm ampicillin, wherein the staring yeastconcentration is 0.1 (OD600), and fermentation is carried out at 48 hrsat 30° C. with agitation.
 4. The engineered yeast of claim 1, whereinthe glucoamylase comprises a sequence having 85% or greater sequenceidentity to SEQ ID NO:1.
 5. The engineered yeast of claim 4, wherein theglucoamylase comprises a sequence having 90% or greater sequenceidentity to SEQ ID NO:1.
 6. The engineered yeast of claim 5, wherein theglucoamylase comprises a sequence having 95% or greater sequenceidentity to SEQ ID NO:1.
 7. The engineered yeast of claim 1 whereinthere are 2-8 copies of the exogenous nucleic acid in the cell.
 8. Theengineered yeast of claim 1 which is a Saccharomyces cerevisiae yeast.9. The engineered yeast of claim 1 which is tolerant to growth in afermentation medium having a concentration of ethanol of greater than 90g/L.
 10. The engineered yeast of claim 1 which is tolerant to growth attemperatures in the range of greater than 31° C.-35° C.
 11. Afermentation method for producing a bioproduct, comprising: forming afermentation medium from a glucose polymer-containing feedstock; andfermenting the fermentation medium using an engineered yeast comprisingan exogenous nucleic acid encoding a glucoamylase comprising a sequencehaving 81% or greater sequence identity to SEQ ID NO:1, whereinfermenting produces a bioproduct.
 12. The fermentation method of claim11, wherein the glucose polymer-containing feedstock or the fermentationmedium, at the beginning of fermentation, has a DE of about 50 or less.13. The fermentation method of claim 11 wherein the glucosepolymer-containing feedstock comprises glucose polymer having a degreeof polymerization of 4 or greater and present in an amount of 75% weightor greater total fermentable carbohydrates in the feedstock.
 14. Thefermentation of any of claim 11 wherein ethanol is produced to aconcentration of 70 g/L or greater in the medium.
 15. The fermentationmethod of any of claim 11 comprising adding supplemental glucoamylase tothe feedstock, or supplemental glucoamylase to the medium during thefermentation period.